Anti-p-selectin antibodies and methods of their use and identification

ABSTRACT

Antibodies are disclosed which bind specifically to P-selectin and which block the binding of PSGL-1 to P-selectin. These anti-P-selectin antibodies may also cause dissociation of preformed P-selectin/PSGL-1 complexes. The disclosure identifies a heretofore unrecognized, near N-terminal, antibody binding domain (a conformational epitope) of P-selectin to which the function-blocking antibodies (which may be chimeric, human or humanized antibodies for example) bind. Antibodies are disclosed which bind to the conformational epitope of P-selectin and which have a dual function in blocking binding of PSGL-1 to P-selectin, and in causing dissociation of preformed P-selectin/PSGL-1 complexes. Such single and dual function anti-P-selectin antibodies and binding fragments thereof may be used in the treatment of a variety of inflammatory and thrombotic disorders and conditions. Screening methods for identifying such antibodies are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 12/516,987,filed May 29, 2009, which claims priority under 35 U.S.C. 371 fromInternational Application No. PCT/US2007/024692, filed Nov. 30, 2007,which claims benefit of U.S. Provisional Application No. 60/872,170,filed Dec. 1, 2006. Each of the above applications is hereby expresslyincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND

The present invention relates to antibodies and antibody fragments whichbind to specific conformational epitopes of P-selectin, and to methodsof their use and identification.

In normal hemostasis and immune surveillance, leukocytes circulatefreely in the blood and respond to injury and infection in a sequentialprocess of adhesion signaling mediated by cell adhesion molecules (1-3).In inflammatory and thrombotic disease, this process is dysregulated andcan sustain pathology wherein leukocytes attack the body's own tissueand can cause serious and sometimes deadly complications. It is wellknown that leukocyte adhesion plays a major role in the pathology ofmany inflammatory and thrombotic disorders such as vasoocclusion insickle cell disease, reperfusion injury, thrombosis, atherosclerosis,asthma, rheumatoid arthritis, psoriasis and tumor metastasis (4-15) deepvenous thrombosis (DVT). P-selectin is also involved in other diseaseprocesses, such as tissue and organ damage associated with inflammation,e.g., ischemia-reperfusion injury. P-selectin is thus a target forintervention in human inflammatory and thrombotic diseases.

Selectins are a family of adhesion proteins which are known to play keyroles in the recruitment of leukocytes to activated endothelium andplatelets. P-selectin is a member of the selectin family of adhesionglycoproteins which also includes L- and E-selectins (16). The selectinsmediate the recruitment, initial tethering and rolling, and adherence ofleukocytes to sites of inflammation (1). P-selectin is stored inWeibel-Palade bodies of endothelial cells and alpha-granules ofplatelets and is rapidly mobilized to the plasma membrane uponstimulation by vasoactive substances such as histamine and thrombin(17).

Sickle Cell Disease

Sickle cell disease (SCD) is a rare inherited blood disorder that causeschronic anemia and vasoocclusion, affecting primarily people ofAfrican-American heritage in the United States. Sickle cell disease isthe most common single gene disorder in African Americans, affectingapproximately 1 in 375-600 persons of African ancestry (18, 19). Sicklecell conditions are also common among people of Mediterranean countries,Africa the Caribbean and parts of South and Central America (18, 19).

SCD is an autosomal recessive disease caused by a single missensemutation (Val6Ala) in the β-globin gene that renders the mutanthemoglobin less soluble and prone to polymerization upon deoxygenation.The polymerization of hemoglobin causes deformation of the erythrocyteto give the cell a sickled shape (20).

SCD has three common variants: homozygous sickle cell disease(hemoglobin SS disease), doubly heterozygous sickle hemoglobin C disease(hemoglobin SC disease) and the sickle β-thalassemias. The most commonand severe form of the disease occurs in individuals who inherit twocopies of the HbS variant (HbSS) and the primary hemoglobin in their redblood cells is sickle hemoglobin. Other individuals can be affected ascompound heterozygotes with varying severity of the disease. They haveone copy of the HbS variant paired with a copy of another β-globin genevariant. HBSC results in a mild form of the disease. Hb β-thalassemiavariants (resulting in the inability to produce the normal βA globinchain)(β° or a reduction in its production ((β⁺) result in a range ofclinical severities. HbS β° is a severe form, whereas HbS β⁺ can bemoderate or mild based on the contribution of each variant to the totalhemoglobin of the patient. Other more rare variants can result if inconjunction with the S gene, another abnormal hemoglobin is inheritedfrom the other parent, such as D, G or O. The predominant form of sicklecell is present in individuals with one copy of HbS and one copy of thenormal β-globin gene (HbA). These individuals carry the sickle celltrait (18).

SCD affects an estimated 50-100,000 people in the US (21-24). Allindividuals that are homozygous or compound heterozygous for HbS showsome clinical manifestations of SCD. Symptoms usually appear within thefirst 6 months of life but there is considerable variability in SCDseverity (25). Individuals with HbSS are most severely affected,followed by individuals with HBbS β°-thalassemia (22, 26). In additionto genotype, additional factors affect disease severity such as thelevels of fetal hemoglobin and the haplotye of the β-globin cluster, aregion that contains 5 genes that code for the β portion of hemoglobin.Despite the capacity to determine genetic risk factors, the ability topredict disease course from birth is limited (27).

In the USA, sickle cell screening at birth is mandated in all 50 statesand the District of Columbia (28) and offers an opportunity for earlyintervention. Diagnostic testing methodology usually comprises acomplete blood count in conjunction with one or more of hemoglobinelectrophoresis, isoelectric focusing, high-performance liquidchromatography and DNA testing (22).

Chronic Anemia and Hemolysis

The sickled erythrocyte has a shorter half-life than the normalerythrocyte and results from the instability of HbS and the effects ofrepeated episodes of hemoglobin polymerization/depolymerization in thecirculation. This affects membrane ionic permeability, cellularviscosity and deformability (20) and promotes oxidative membrane damage(29). Sickle cell disease patients are anemic by 2 to 3 months of ageand develop symptoms and complications associated with chronic anemiaand hemolysis (22, 30) such as renal disease, ophthalmic disorders, legulcers, priapism and pulmonary hypertension (26, 31-37). Hemoglobinvalues for SCD patients range from 6 to 10 g/dL and the hemoglobin Smolecule has a poor affinity for oxygen. Triggers for transfusion inpatients are a hemoglobin value of 5 or less or a precipitous drop inhemoglobin of 2 g/dL or more. Transfusions are typically given torestore hemoglobin values to baseline levels established for eachpatient as excessive hemotocrit can precipitate sickling (38). SCDpatients are more susceptible to parvovirus B19 infection which canarrest erythropoiesis and lead to aplastic anemia crisis (39).

Vasoocclusive Pain Crisis

Vascular occlusion is central to the clinical course of SCD and likelyinvolves both the micro and macro circulation. Occlusion occurring inthe microvasculature can culminate in acute painful episodes orvasoocclusive pain crises. Vasoocclusive pain crisis is the clinicalhallmark of microvascular occlusions and accounts for over 90% ofhospital admissions of adults SCD patients. It is well known thatpolymerization of hemoglobin S during deoxygenation and cell sicklingleads to blockage of the microvasculature (40). However, it has recentlybecome clear that hemoglobin S polymerization is not solely responsiblefor vasoocclusion. It has now been demonstrated that such events assickled red cell lysis, cell membrane damage and oxidative stress,repeated ischemic damage, and microvasculature injury due to theadhesive interactions between sickle red cells and the endothelium thatculminate in a proinflammatory environment (41-43). In this environmentof chronic vascular inflammation, the adherence of leukocytes, plateletsand sickled red cells to activated blood vessel endothelium and to eachother is believed to be a primary cause of microvasculature blockage andvasoocclusive pain crisis (43-47). Additional factors such as therigidity of sickled cells, increased blood viscosity, and localvasoconstriction have also been identified as potentially contributingto the vasoocclusion process.

Long-term repeated vasoocclusive events and occlusions occurring in themacrovasculature can cause life-threatening complications leading toorgan damage and failure, stroke and death (40). There is anapproximately 20 to 30 year reduction in life expectancy in sickle celldisease patients (48). Chronic pain in SCD is not just a continuation ofthe pain of vasoocclusion: it is usually secondary to avascular necrosisof bone at various joints (49). Sickled red cells can become trapped inthe spleen causing it to become enlarged and precipitating splenicsequestration crisis causing sudden and severe anemia. Functionalasplenia leaves patients susceptible to infection (18). Bone growthretardation, renal (32), ophthalmic (33) and cerebrovascularcomplications (ranging from clinically evident acute stroke to transientsilent ischemic infarct) (50) are seen as major clinical consequences ofSCD and vasoocculsive injury (22). Acute chest syndrome is another majorcomplication (51), and is a significant cause of morbidity and mortality(52).

Pain episodes appear to be triggered by a number of factors includingcold, stress and physical exertion (38, 53). The frequency, severity,location and duration of pain crises can vary considerably, even withina specific disease subtype. Patients with homozygous sickle cell andsickle cell β°-thalassemia have a higher frequency of vasoocclusive paincrises than patients with hemoglobin SC and sickle cell-β°-thalassemiagenotype (54). Disease severity is thought to depend on a complexinteraction of genetic, rheologic and hematologic factors, as well asmicrovascular and endothelial factors. Crises commonly involve pain inthe back, legs, knees, arms, chest and abdomen (53). The frequency ofcrisis and pain severity varies considerably among patients and in thesame patient over time. One study evaluating pain rates in patientsranging from newborns to age 50 years indicated that 5.2 percent ofpatients with sickle cell disease have three to 10 episodes of severepain every year (54). In an independent study, over 30% of sickle cellpatients in the US (approximately 27,000 patients) have three or morepain crises per year (55). Moreover, a recent study (PISCES) evaluatinghealth related quality of life issues in SCD patients indicated thatpain crisis might be significantly underreported among SCD patients(56).

Current Therapies For Vascular Occlusion

Vascular occlusion in SCD patients can manifest in multiple waysincluding vasoocclusive pain crisis, acute chest syndrome,cerebrovascular events and multiple types of organ failure. Therefore,treatment modalities for vascular occlusion depend on the clinicalcourse and severity of the disease and are generally symptomatic orpalliative in nature. Patient education in the avoidance of initiatingfactors that precipitate vasoocclusive pain crisis has shown someprophylactic benefit. The two most common symptomatic treatments areblood transfusions and analgesics. Most SCD patients commonly havehemoglobin values between 6 and 10 g/dL and hemoglobin values typicallydrop at least 1 g per dL during a vasoocclusive pain crisis. Severe painresulting from vasoocclusive crisis can be treated with narcotics buttheir use is controversial due to concerns of narcotic addiction andtolerance. Other complications with narcotic use are drug-seekingbehavior, sedation and respiratory depression. Oxygen management hasbeen utilized to treat vasoocclusive pain crisis despite the lack ofstrong evidence supporting its effectiveness. Rehydration is alsosometime used during vasoocclusive pain crises with some benefit (22,38).

Bone marrow transplantation may be considered and can be curative, butits use is restricted to a limited number of patients, and carries ahigh risk of morbidity and mortality (22).

Hydroxyurea (Droxia) is the only FDA approved drug for treatment of SCDpain crises. The mechanisms by which it produces its beneficial effectsare uncertain but may involve increasing hemoglobin F levels in RBCsthereby decreasing the level of hemoglobin S polymerization. Hydroxyureais cytotoxic, myelosuppressive and teratogenic (57, 58) which implies acarcinogenic risk to SCD patients. The long-term effects however, onhematologic toxicities, organ damage and carcinogenicity are currentlyunknown (59, 60).

In summary, most therapies for vasoocclusive pain crisis in SCD patientsprovide symptomatic relief and do not address the underlying cause ofthis debilitating condition. To date only one therapy has been approvedby the FDA for the treatment of pain crisis, thus, patients with SCDrepresent a major unmet medical need in a life-threatening disease withsevere morbidities.

P-selectin as a Therapeutic Target for SCD

In SCD, as noted above, interactions between sickled red cells,platelets, leukocytes and the microvasculature are P-selectin-dependentprocesses and result in vasoocclusion and painful crisis. Studies intransgenic mice engineered to express human β hemoglobin S (β^(S)) haveshown that antibody-mediated inhibition of P-selectin function canprevent and/or reduce vasoocclusion, indicating a therapeutic potentialfor this target. In addition mice expressing the β^(S) hemoglobin thatlack P-selectin (due to gene deletion) do not suffer vasoocclusion,further supporting a key role for this molecule in this morbidity.

The hyper-inflammatory state in SCD patients is characterized byactivated monocytes and vascular endothelium (61-63). A similarpro-inflammatory phenotype was demonstrated in resting state β^(S) micewhich exhibit elevated levels of peripheral leukocytes and neutrophils,an increased number of rolling and adherent leukocytes, and reducedblood flow volume and red blood cell velocities (64). The β^(S) micewere hypersensitive to hypoxia/reoxygenation resulting in aninflammatory response represented by a significant increase in thenumber of adherent and emigrated leukocytes. This inflammatory responsewas completely blocked by a functionally blocking anti-mouse P-selectinantibody, but not by a functionally blocking anti-mouse E-selectinantibody, demonstrating a critical role for P-selectin in this process.

Inflammatory Bowel Disease

Inflammatory Bowel Disease (“IBD”) is the collective term used todescribe two chronic, idiopathic inflammatory diseases of thegastrointestinal tract: ulcerative colitis (“UC”) and Crohn's Disease(“CD”). UC and CD are considered together because of their overlappingclinical, etiologic, and pathogenetic features. From a therapeutic andprognostic standpoint, however, it is useful to distinguish them.

IBD occurs world-wide and is reported to afflict as many as two millionpeople. Onset has been documented at all ages; however, IBDpredominately begins in young adulthood. The three most commonpresenting symptoms of IBD are diarrhea, abdominal pain, and fever. Thediarrhea may range from mild to severe and is often accompanied byurgency and frequency. In UC, the diarrhea is usually bloody and maycontain mucus and purulent matter as well. Anemia and weight loss areadditional common signs of IBD. Reports of an increasing occurrence ofpsychological problems, including anxiety and depression, are perhapsnot surprising secondary effects of what is often a debilitating diseasethat occurs in people in the prime of life.

A battery of laboratory, radiological, and endoscopic evaluations arecombined to derive a diagnosis of IBD and to assess the extent andseverity of the disease. Nevertheless, differentiating UC from CD, aswell as other types of inflammatory conditions of the intestines, suchas irritable bowel syndrome, infectious diarrhea, rectal bleeding,radiation colitis, and the like, is difficult, because the mucosa of thesmall and large intestines reacts in a similar way to a large number ofdifferent insults. Once other types of bowel disorders have been ruledout, the final diagnosis is often made on the basis of the progressionof the disease. In many patients, though, the colitis must still beregarded as indeterminate because of the overlapping features of UC andCD, particularly with CD of the colon.

The leading early symptoms of UC and CD are chronic recurrent diarrhea,bloody diarrhea, recurrent abdominal pain, nausea, weight loss generalevidence of inflammation without any obvious explanation (fever, raisedESR, leucocytosis, thrombocytosis and dysproteinenemia or anemia). Amongthese symptoms, diarrhea and anemia are more characteristic of UC whilepain and weight loss and marked evidence of inflammation are more commonin CD. While the history and physical examination of a patient can help,the final confirmation of the diagnosis has traditionally been madeendoscopically, histologically and, in relation to the small intestine,radiologically as well.

The SAMP-1/Yit mouse model of spontaneous iletis closely resembles humanCrohn's Disease (65, 66). Therapeutic inhibition of PSGL-1 bindinguniquely ameliorates ileitis in this model whereas blockade ofindividual selectins does not (67). Inhibition of TNF in this model doesreduce the severity of ileitis in a manner similar to anti-PSGL-1binding although the therapeutic effect does not appear to be as potentas anti-PSGL-1. Thus, the SAMP-1 model appears to closely mirror humanCrohn's Disease not only in its pathophysiology but also in its responseto therapeutic intervention. This evidence points to the conclusion thattherapeutic substances which inhibit P-selectin-PSGL-1 binding activityin humans (and other primates) would also be effective as treatments ofCrohn's Disease.

P-selectin plays its central role in the recruitment of leukocytes toinflammatory and thrombotic sites by binding to its counter-receptor,P-selectin glycoprotein ligand-1 (PSGL-1) (or a PSGL-1-like receptor onsickled red blood cells), which is a mucin-like glycoproteinconstitutively expressed on leukocytes including neutrophils, monocytes,platelets, and on some endothelial cells (68). The ultimate physiologicfunction of the selectins is to promote extravasation of leukocytes intoinflamed or damaged tissues. The initial binding of P-selectin on theendothelium to PSGL-1 on the leukocytes is essential and central to thisprocess. The predominant mechanism for rolling and tethering ofleukocytes to activated endothelium and platelets is the binding ofleukocyte PSGL-1 to the P-selectin on these cells (68, 69). PSGL-1 bindsto P—, L- and E-selectin (70). P-selectin and SGP-3, a glycosulfopeptidemodeled from the N-terminus of PSGL-1, have been co-crystallized and thecontact residues for lectin-ligand binding have been identified (71).

The selectins share common structural motifs including a lectin domain(or carbohydrate recognition domain), an epidermal growth factor-likedomain (EGF), a varying series of consensus repeats, a transmembranedomain and a cytoplasmic tail (70). As noted, the initial tethering androlling of leukocytes is mediated by the interaction of P-selectin andPSGL-1. Thus the blocking of P-selectin function by using (1) antibodiesto P-selectin, (2) antibodies to PSGL-1, (3) fragments of PSGL-1 orrecombinant forms of PSGL-1, (4) small molecules that mimic the bindingdomain of PSGL-1, and (5) other molecules that disrupt the binding ofP-selectin to PSGL-1, can block leukocyte rolling and tethering and thusprevent firm adhesion to endothelial cells or platelets. Mice deficientin P-selectin or PSGL-1 also fail to support leukocyte tethering androlling on activated endothelial cells (72, 74). L-selectin plays a dualrole in that it is constitutively expressed on circulating leukocytesand can initiate “secondary binding” by interaction with PSGL-1 on otherleukocytes (75). This process leads to further recruitment of newleukocytes to the inflamed area. L-selectin binding to PSGL-1 also playsa role in homing of lymphocytes to the high endothelial vasculature(HEV) venules in the secondary lymphatic system (76). E-selectin istranscriptionally regulated and is expressed on activated endothelialcells hours after P-selectin mediated events. E-selectin can bind PSGL-1with low affinity but can also bind other ligands. Single transgenicknockout mice for each selectin have shown that these molecules possesscompensatory selectin mechanisms for leukocyte homing and rolling (77).

In view of the above, there is a well-established need for newtreatments, such as antibodies, that target P-selectin as a means oftreating inflammatory and thrombotic diseases by disrupting the bindingof P-selectin and PSGL-1. It is therefore a preferred goal of thepresent invention to block P-selectin binding to PSGL-1 to block theadherence of blood cells that contribute to vasoocclusion in SCD andother thrombotic disorders.

SUMMARY OF THE DISCLOSURE

The presently disclosed and claimed inventive concepts, in oneembodiment, are directed to “function-blocking” antibodies which bindspecifically to P-selectin and which block the binding of PSGL-1 toP-selectin. These anti-P-selectin antibodies may also cause dissociationof preformed P-selectin/PSGL-1 complexes. The present disclosuredescribes a heretofore unrecognized antibody binding domain (aconformational epitope) within the lectin domain (e.g., carbohydraterecognition domain, CRD) of P-selectin to which the function-blockingantibodies (which may be chimeric, human or humanized antibodies orfragments thereof for example) bind. The presently disclosed and claimedinventive concepts, in one embodiment, are also directed toanti-P-selectin antibodies which bind to the conformational epitopedescribed herein and which have a dual function in (1) blocking bindingof PSGL-1 to P-selectin, and (2) causing dissociation of preformedP-selectin/PSGL-1 complexes. The presently disclosed and claimedinventive concepts in particular are directed to using such single anddual function anti-P-selectin antibodies or antibody fragments asdescribed herein in treatments for inflammatory, thrombotic or otherconditions or disorders in primates (including humans) which involveplatelet, sickled red cell, leukocyte, lymphocyte, and/or endothelialcell adhesion, wherein the condition or disorder comprises or isassociated with (but not limited to) at least one of sickle cellvasoocclusive pain crisis, inflammatory bowel disease (e.g., Crohn'sDisease, ulcerative colitis, enteritis), arthritis (e.g., rheumatoidarthritis, osteoarthritis, psoriatic arthritis), graft rejection, graftversus host disease, asthma, chronic obstructive pulmonary disease,psoriasis, dermatitis, sepsis, nephritis, lupus erythematosis,scleroderma, rhinitis, anaphylaxis, diabetes, multiple sclerosis,atherosclerosis, thrombosis, tumor metastasis, allergic reactions,thyroiditis, ischemic reperfusion injury (e.g., due to myocardialinfarction, stroke, or organ transplantation), and conditions associatedwith extensive trauma, or chronic inflammation, such as for example,type IV delayed hypersensitivity, associated for example with infectionby Tubercle bacilli, or systematic inflammatory response syndrome, ormultiple organ failure. Other embodiments of the inventive conceptsdisclosed and claimed herein will be apparent in the DetailedDescription below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a homology comparison at the amino acid level of human andmouse P-selectin indicating the location of lectin, EGF, CR1 and CR2domains (transition between domains is indicated by ↓). Nonlinearconformational domains. A, B, C1, D, E1, C2, E2, C3, and F are indicatedby dashed boxes. Amino acid differences are indicated in boldface.

FIG. 2 shows a representative sensogram showing the ability of the G1test antibody to bind various P-selectin chimeras: SEQ ID NO:1 (humanP-selectin), SEQ ID NO:2 (mouse P-selectin), and SEQ ID NO:4 (humancluster A (aa 1-35) replaces mouse aa 1-35 in mouse P-selectin). The G1antibody binds to human P-selectin (SEQ ID NO:1), does not bind to mouseP-selectin (SEQ ID NO:2), and binds mouse P-selectin when human aminoacids from Cluster A have been inserted into the mouse sequence inresidues 3-23 (SEQ ID NO:4).

FIG. 3 shows representative two-step BIACORE P-selectin chimera bindingdata for G1, G3, G4 and G5 anti-P-selectin antibodies binding to SEQ IDNO:1-4, 7-10, 18 and 19. G4 was an uncharacterized anti-P-selectinantibody and demonstrated P-selectin binding properties similar to G1.

FIG. 4 shows BIACORE sensograms demonstrating dissociation of thepreformed P-selectin/PSGL-1 complex upon exposure to dual functionanti-P-selectin antibodies (G1, G4, hSEL001). PSGL-1 is represented byGSP-6 peptide, a PSGL-1 mimetic. Initial RU increase shows binding ofP-selectin to biotin-GSP-6 coupled to a streptavidin coated BIACOREchip. Once steady state binding of the P-selectin/GSP-6 complex wasreached (i.e., after normal dissociation of the complex had reachednear-equilibrium), test antibodies G1, G3, and G5 were injected andassessed for dissociation properties. G1 caused dissociation of thepreformed P-selectin/GSP-6 complex. G5 bound to the preformed complex,but did not cause its dissociation. G3 did not bind or dissociate thepreformed P-selectin/PSGL-1 complex. A new anti-P-selectin antibody, G4,and a humanized anti-P-selectin antibody named hSEL001, also both boundand caused dissociation of the preformed P-selectin/PSGL-1 complex,indicating dual function capabilities.

FIG. 5 shows a 3-D representation of a human P-selectin molecule withGSP-6 binding thereto. Lectin and EGF domains are demarcated by a dashedline. Binding region 1 indentifies a Cluster A conformational epitopethat is distal to the lectin/ligand binding domain. Test antibody G1bound to region 1, Cluster A. G4, and a humanized anti-P-selectinantibody, hSEL001, also bound region 1, Cluster A.

FIG. 6 shows graphs of results of cell-based in vitro rolling assaysunder flow of human neutrophils on low (A) and high (B) densityP-selectin. Results demonstrate blocking and/or dissociation of thepreformed P-selectin/PSGL-1 complex and subsequent release ofneutrophils upon exposure to antibodies G1, G3 and hSel001. Antibodieswere introduced at equivalent concentrations of 20 μg/ml for theduration of the study. There is a lag time of about 1 minute before theantibody reaches the chamber due to the dead volume of the system. At1-minute intervals thereafter, cells remaining bound were counted andexpressed as % cells bound. Panel (A) shows neutrophils rolling ataverage velocity of 5 μm/s on low density (50 sites/μm²) membraneP-selectin. Panel (B) shows neutrophils rolling at an average velocityof 1 μm/s on high density P-selectin (380 sites/μm²).

DETAILED DESCRIPTION

As indicated above, the presently disclosed and claimed inventiveconcepts, in one embodiment, are directed to antibodies which bindspecifically to P-selectin and which block the binding of PSGL-1 toP-selectin (and are referred to herein as “function-blocking”antibodies). In some embodiments, these anti-P-selectin antibodies mayalso cause dissociation of preformed P-selectin/PSGL-1 complexes. Thedisclosure describes a heretofore unrecognized antibody binding domain(a conformational epitope) within the lectin domain (e.g., carbohydraterecognition domain, CRD) of P-selectin to which the function-blockingantibodies (which may be chimeric, human or humanized antibodies, orfragments thereof for example) bind. The presently disclosed and claimedinventive concepts also directed to anti-P-selectin antibodies whichbind to the conformational epitope described herein and which have adual function in (1) blocking binding of PSGL-1 to P-selectin and (2)causing dissociation of preformed P-selectin/PSGL-1 complexes. Thepresently disclosed and claimed inventive concepts in particular aredirected to using such single and dual function anti-P-selectinantibodies or antibody fragments as described herein in treatments forinflammatory, thrombotic or other conditions or disorders in primates(including humans) which involve platelet, sickled red cell, leukocyte,lymphocyte, and/or endothelial cell adhesion, wherein the condition ordisorder comprises or is associated with (but not limited to) at leastone of sickle cell vasoocclusive pain crisis, inflammatory bowel disease(e.g., Crohn's Disease, ulcerative colitis, enteritis), arthritis (e.g.,rheumatoid arthritis, osteoarthritis, psoriatic arthritis), graftrejection, graft versus host disease, asthma, chronic obstructivepulmonary disease, psoriasis, dermatitis, sepsis, nephritis, lupuserythematosis, scleroderma, rhinitis, anaphylaxis, diabetes, multiplesclerosis, atherosclerosis, thrombosis, tumor metastasis, allergicreactions, thyroiditis, ischemic reperfusion injury (e.g., due tomyocardial infarction, stroke, or organ transplantation), and conditionsassociated with extensive trauma, or chronic inflammation, such as forexample, type IV delayed hypersensitivity, associated for example withinfection by Tubercle bacilli, or systematic inflammatory responsesyndrome, or multiple organ failure. The presently disclosed and claimedinventive concepts are also directed to a screening assay for thedetection of anti-P-selectin antibodies which bind to the conformationalepitope of P-selectin described here, and, in one embodiment, also blockbinding of PSGL-1 to P-selectin, and which, in another embodiment, causereversal of the binding of PSGL-1 to P-selectin (i.e., dissociation ofthe preformed complex).

It is noted that as used herein and in the appended claims, the singularforms “a”, “an”, and “the” include plural reference unless the contextclearly dictates otherwise. Thus, for example, reference to “a cell”includes a plurality of such cells and equivalents thereof known tothose skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising”, “including”,“characterized by” and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the presently disclosed and claimed inventiveconcepts, the preferred methods and materials are now described.

“Leukocyte rolling,” as used herein, includes weak adhesion ofleukocytes to endothelial cells of blood vessels and rolling ofleukocytes along endothelial cells of blood vessels prior to firmadhesion and transmigration of leukocytes into endothelial tissue.Following leukocyte rolling, these adherent leukocytes can migratethrough the endothelium and destroy ischemic tissue during reperfusion.Accordingly, reduction of leukocyte rolling results in a reduction ofdamage to tissues and organs caused by acute inflammatory responses.

As used herein, a “P-selectin antagonist” includes any agent which iscapable of antagonizing P-selectin, e.g., by inhibiting interactionbetween P-selectin and a P-selectin glycoprotein ligand-1, e.g., byinhibiting interactions of P-selectin expressing endothelial cells andactivated platelets with PSGL-1 expressing leukocytes.

As noted herein, the presently disclosed and claimed inventive conceptsare directed to purified antibodies (including but not limited tochimeric, human, or humanized antibodies and fragments thereof), whichrecognize (i.e., bind to) P-selectin (SEQ ID NO:1) and which blockbinding of P-selectin to PSGL-1 (or PSGL-1-like receptors), and tomethods for screening for such antibodies and binding fragments thereof,and to therapeutic methods of use thereof.

More particularly, the presently disclosed and claimed inventiveconcepts are directed to purified antibodies (or fragments thereof),against P-selectin, host cells that produce such anti-P-selectinantibodies (or fragments thereof), screening assays to identifyanti-P-selectin antibodies (or fragments thereof) which blockleukocytes, sickled erythrocytes, lymphocyte, platelet and endothelialcell P-selectin-mediated adhesion and optionally further causedissociation of preformed adhesions or cell complexes that were mediatedthrough PSGL-1/P-selectin interactions, and therapeutic methods usingsuch antibodies (or binding fragments thereof). The presently disclosedand claimed inventive concepts include novel antibodies against primate(including human) P-selectin and binding fragments thereof, particularlyincluding, in one embodiment, hSel001 antibody. Preferred antibodies ofthe disclosure are capable of specifically binding primate (particularlyhuman) P-selectin, and inhibiting one or more P-selectin activities invitro and/or in vivo. Where used herein, the term “PSGL-1” is alsointended to include “PSGL-1-like receptor” on sickled red cells(erythrocytes).

Antibodies

Antibody molecules belong to a family of plasma proteins calledimmunoglobulins, whose basic building block, the immunoglobulin fold ordomain, is used in various forms in many molecules of the immune systemand other biological recognition systems. A typical immunoglobulin hasfour polypeptide chains, containing an antigen binding region known as avariable region and a non-varying region known as the constant region.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end. The constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG₁, IgG₂, IgG₃ and IgG₄ and IgA₁ and IgA₂. Theconstant domains of the heavy chains that correspond to the differentclasses of immunoglobulins are called alpha (α), delta (δ), epsilon (ε),gamma (γ) and mu (μ), respectively. The light chains of antibodies canbe assigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino sequences of their constant domain. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies. The variable domains are forbinding and determine the specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments per chain called complementaritydetermining regions (CDRs), also known as hypervariable regions, both inthe light chain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four FR regions, largely adopting a (3-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the 13-sheet structure. The CDRs in eachlight and heavy chain are held together in close proximity by the FRregions and contribute to the formation of the antigen-binding site ofthe antibody. The constant domains are not involved directly in bindingan antibody to an antigen, but exhibit various effector functions, suchas participation of the antibody in antibody-dependent cellulartoxicity.

An antibody of the presently disclosed and claimed inventive conceptsthus can be in any of a variety of forms, including a wholeimmunoglobulin, an antibody fragment such as Fv, Fab, and similarfragments, a single chain antibody which includes the variable domaincomplementarity determining regions (CDRs), and the like forms, all ofwhich fall under the broad term “antibody”, as used herein. In preferredembodiments, in the context of both the therapeutic and screeningmethods described below, an antibody or fragment thereof is used that isimmuno-specific for an antigen or epitope of the presently disclosed andclaimed inventive concepts as described herein.

The term “antibody fragment” as used herein refers to a portion of afull-length antibody, generally the antigen binding or variable region.Examples of antibody fragments include Fab, Fab′, F(ab′)₂ and Fvfragments. Papain digestion of antibodies produces two identical antigenbinding fragments, called the Fab fragment, each with a single antigenbinding site, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding fragments that are capable of cross-linkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom anti-body fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

Antibody fragments may be as small as about 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30, 35, 40, 45 or 50 or 75 or 100 (inclusive) or more amino acids,for example. In general, an antibody fragment of the presently disclosedand claimed inventive concepts can have any upper size limit so long asit is has similar or immunological properties relative to antibody thatbinds with specificity to the P-selectin-binding described herein andwhich blocks binding of PSGL-1 to P-selectin. Where used herein the term“inclusive” is intended to refer to all integers between any two numberslisted herein.

As noted elsewhere herein, antibody fragments contemplated herein retainthe ability to selectively bind to all of or a portion of the P-selectinbinding epitope described herein. Preferably, an antibody or bindingfragment of an antibody of the present invention is capable of bindingto an epitope comprising one or more of amino acid residues 1-35, or,more particularly, 4-23, of the sequence set forth in SEQ ID NO:1. Sometypes of antibody fragments are defined as follows:

Fab is the fragment that contains a monovalent antigen-binding fragmentof an antibody molecule. A Fab fragment can be produced by digestion ofwhole antibody with the enzyme papain to yield an intact light chain anda portion of one heavy chain.

Fab′ is the fragment of an antibody molecule can be obtained by treatingwhole antibody with pepsin, followed by reduction, to yield an intactlight chain and a portion of the heavy chain. Two Fab′ fragments areobtained per antibody molecule.

Fab′ fragments differ from Fab fragments by the addition of a fewresidues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region.

(Fab′)₂ is the fragment of an antibody that can be obtained by treatingwhole antibody with the enzyme pepsin without subsequent reduction.F(ab′)₂ is a dimer of two Fab′ fragments held together by two disulfidebonds.

Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in a tight, non-covalentassociation (VH-VL dimer). It is in this configuration that the threeCDRs of each variable domain interact to define an antigen binding siteon the surface of the VH-VL dimer. Collectively, the six CDRs conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

A single chain antibody (SCA) is defined herein as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. Such single chainantibodies are also referred to as “single-chain Fv” or “sFv” or “scFv”antibody fragments. Generally, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains that enables the sFv toform the desired structure for antigen binding.

As noted above, the antibodies or antibody fragments of the presentlydisclosed and claimed inventive concepts in preferred embodimentscomprise immunoglobulins of the isotypes IgG₁, IgG₂, IgG₃, IgG₄ orIgG₂/G₄ chimeras, preferably binds to P-selectin with a high affinity(for example wherein the K_(d) is ≦1000 nM) and preferably comprises ahuman constant region, and preferably inhibits binding of P-selectin toPSGL-1 and more preferably also caused reversal of binding of P-selectinto PSGL-1 in a preformed complex. Further, the anti-P-selectin antibodyor binding fragment thereof preferably does not activate complement viathe classical pathway by interacting with C1q and preferably does notbind Fc receptors, collectively called antibody effector function. Thepresently disclosed and claimed inventive concepts in particular aredirected to using such single and dual function anti-P-selectinantibodies or antibody fragments as described and identified herein intreatments for inflammatory, thrombotic or other conditions or disordersin primates (including humans) which involve platelet, sickled red cell,leukocyte, lymphocyte, and/or endothelial cell adhesion, wherein thecondition or disorder comprises or is associated with (but not limitedto) at least one of sickle cell vasoocclusive pain crisis, inflammatorybowel disease (e.g., Crohn's Disease, ulcerative colitis, enteritis),arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriaticarthritis), graft rejection, graft versus host disease, asthma, chronicobstructive pulmonary disease, psoriasis, dermatitis, sepsis, nephritis,lupus erythematosis, scleroderma, rhinitis, anaphylaxis, diabetes,multiple sclerosis, atherosclerosis, thrombosis, tumor metastasis,allergic reactions, thyroiditis, ischemic reperfusion injury (e.g., dueto myocardial infarction, stroke, or organ transplantation), andconditions associated with extensive trauma, or chronic inflammation,such as for example, type IV delayed hypersensitivity, associated forexample with infection by Tubercle bacilli, or systematic inflammatoryresponse syndrome, or multiple organ failure.

As noted elsewhere herein, P-selectin plays a central role inrecruitment of leukocytes and lymphocytes to inflammatory and thromboticsites by binding to a surface ligand (PSGL-1) on these cells and in thebinding of sickled red cells to endothelia having activated endothelialcells. PSGL-1 is constitutively expressed on leukocytes, includingneutrophils and monocytes, and on some endothelial cells. A PSGL-1-likereceptor is expressed on sickled red cells and enables these cells tobind P-selectin on activated endothelial cells.

Without wanting to be bound by theory, it is believed that the treatmentof vasoocclusive sickle cell pain crisis, for example by theanti-P-selectin antibody of the present invention, is effective byinhibiting any one or more of the following interactions: (1) PSGL-1 onleukocytes binding to P-selectin on activated endothelium; (2) aPSGL-1-like ligand on sickled red cells binding to P-selectin onactivated endothelium; (3) P-selectin on the surface of activatedplatelets binding PSGL-1 on endothelial cells; (4) sickled red cellsbinding leukocytes through an uncharacterized ligand-receptorinteraction; (5) P-selectin on activated platelets binding the PSGL-1like receptor on sickled red cells, and/or; (6) P-selectin on thesurface of activated platelets binding PSGL-1 on leukocytes. It isexpected that the function-blocking anti-P-selectin antibody blocks theinitiation and propagation of vasoocclusion at multiple levels ofcell-cell interactions in the microvasculature.

The presently disclosed and claimed inventive concepts in one embodimentare directed to antibodies that specifically bind to human P-selectin.CDRs in such antibodies are not limited to the specific sequences of VHand VL shown herein or in the documents incorporated by reference hereinand may include variants of these sequences that retain the ability toblock P-selectin binding to PSGL-1. Such variants may be produced by askilled artisan using techniques well known in the art. For example,amino acid substitutions, deletions, or additions, can be made in theFRs and/or in the CDRs as described elsewhere herein. While changes inthe FRs are usually designed to improve stability and decreaseimmunogenicity of the antibody, changes in the CDRs are typicallydesigned to increase affinity of the antibody for its target. Variantsof FRs also include naturally occurring immunoglobulin allotypes. Suchaffinity-increasing changes may be determined empirically by routinetechniques that involve altering the CDR and testing the affinityantibody for its target.

For example, conservative amino acid substitutions can be made withinany one of the disclosed CDRs. Various alterations can be made accordingto methods well known to those skilled in the art (78). These includebut are not limited to nucleotide sequences that are altered by thesubstitution of different codons that encode an identical or afunctionally equivalent amino acid residue (“conservativesubstitutions”) within the sequence, thus producing a “silent” change.For example, the nonpolar amino acids which may be conservativelysubstituted include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine. The polar neutral amino acidswhich may be substituted conservatively include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids which may be conservatively substitutedinclude arginine, lysine, and histidine. The negatively charged (acidic)amino acids which may be conservatively substituted include asparticacid and glutamic acid. Substitutes for an amino acid within thesequence may also be selected from other members of the class to whichthe amino acid belongs.

Derivatives and analogs of antibodies of the invention can be producedby various techniques well known in the art, including recombinant andsynthetic methods (79, 80). Antibodies in which CDR sequences differonly insubstantially from those of the variable regions ofanti-P-selectin antibodies such as hSel001, discussed in further detailbelow, are also encompassed within the scope of this invention. As notedabove, typically, an amino acid is substituted by a related amino acidhaving similar charge, hydrophobic, or stereochemical characteristics.Such substitutions would be within the ordinary skills of an artisan.Further, a skilled artisan would appreciate that changes can be made inFRs without adversely affecting the binding properties of an antibody.Changes to FRs include, but are not limited to, humanizing a non-humanderived or engineering certain framework residues that are important forantigen contact or for stabilizing the binding site, e.g., changing theclass or subclass of the constant region, changing specific amino acidresidues which might alter the effector function such as Fc receptorbinding.

As used herein, the “affinity” of the antibody for P-selectin or theconformational epitope thereof is characterized by its K_(d), ordisassociation constant. A stronger affinity is represented by a lowerK_(d) while a weaker affinity is represented by a higher K_(d). As such,an antibody of the present invention preferably has an affinity for aP-selectin conformational epitope represented by a K_(d)≦1000 nM, or≦500 nM, or ≦100 nM, or ≦50 nM, or more preferably by a K_(d)≦25 nM, andstill more preferably by a K_(d)≦10 nM, and even more preferably by aK_(d)≦5 nM, ≦1 nM, or ≦0.1 nM.

An antibody or antibody fragment “homolog,” as used herein, means that arelevant amino acid sequence (preferably for example in the CDRs and/orvariable domains VH and/or VL) of a protein or a peptide is at least50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or100% identical to a given sequence. By way of example, such sequencesmay be variants derived from various species, or the homologous sequencemay be recombinantly produced. The sequence may be derived from thegiven sequence by truncation, deletion, amino acid substitution, oraddition. Percent identity between two amino acid sequences isdetermined by standard alignment algorithms such as, for example, BasicLocal Alignment Tool (BLAST) and other alignment algorithms and methodsof the art (81-84).

The term “isolated” or “purified” refers to a molecule that issubstantially free of its natural environment. For instance, an isolatedprotein is substantially free of cellular material or other proteinsfrom the cell or tissue source from which it was derived. The term alsorefers to preparations where the isolated protein is at least 70-80%(w/w) pure; or at least 80-90% (w/w) pure; or at least 90-95% pure; orat least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure. Insome embodiments, the isolated molecule is sufficiently pure forpharmaceutical compositions.

Inhibitory activity refers to a reduction in an activity of P-selectinby a P-selectin inhibitor (such as an antibody or fragment thereof),relative to the activity of P-selectin in the absence of the sameinhibitor. A neutralizing antibody may reduce one or more P-selectinactivities. For example, the reduction in activity (e.g., P-selectinbinding to PSGL-1) is preferably at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, or higher. In another example, thedissociative activity of a dual function antibody or fragment (i.e., thepercentage of preformed P-selectin/PSGL-1 complex which may be caused todissociate) may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, or higher.

The term “P-selectin inhibitor” when used herein includes any agent,such as, e.g., a neutralizing antibody, capable of inhibiting activity,expression, processing, binding, or cell surface localization ofP-selectin. Such inhibitors are said to “inhibit,” “neutralize,” or“reduce” the biological activity of P-selectin.

The preparation of monoclonal antibodies is conventional and well knownto persons of ordinary skill in the art. Monoclonal antibodies can beisolated and purified from hybridoma cultures by a variety ofwell-established techniques. Such isolation techniques include affinitychromatography with Protein-A Sepharose, size-exclusion chromatography,and ion-exchange chromatography.

Methods of in vitro and in vivo manipulation of monoclonal antibodiesare well known to those skilled in the art. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein(85), or may be made by recombinant methods, e.g., as described in U.S.Pat. No. 4,816,567, for example.

Another method involves humanizing a monoclonal antibody by recombinantmeans to generate antibodies containing, for example, human or primatespecific and recognizable sequences.

Methods of making antibodies of the presently disclosed and claimedinventive concepts which bind with high affinity to human P-selectin orto the conformational epitopes thereof as described herein may comprisetransfecting a cell with a DNA construct, the construct comprising a DNAsequence encoding at least a portion of the neutralizing P-selectinspecific antibodies of the invention, culturing the cell underconditions such that the antibody protein is expressed by the cell, andisolating the antibody protein.

Preferably, the constant region has been modified to modulate (i.e.reduce or enhance) effector function as noted elsewhere as compared tothe effector function of a wild-type immunoglobulin heavy chain Fcregion. In various embodiments, the IgG constant region has reducedeffector function, or alternatively it has increased effector function,for example. Fc effector function includes, for example,antibody-dependent cellular cytotoxicity (ADCC), phagocytosis,complement-dependent cytotoxicity, and half-life or clearance ratefunction. The IgG amino acid sequence of the Fc domain can be altered toaffect binding to Fc gamma receptors (and thus ADCC or phagocytosisfunctions), or to alter interaction with the complement system(complement-dependent cytotoxicity function).

In one embodiment, the antibody comprises a constant region or Fcportion that has low or no affinity for at least one Fc receptor. In analternative embodiment, the second polypeptide has low or no affinityfor complement protein C1q. In general, an effector function of anantibody can be altered by altering the affinity of the antibody for aneffector molecule such as an Fc receptor. Binding affinity willgenerally be varied by modifying the effector molecule binding site.Disclosure of IgG modifications that alter interaction with effectormolecules such as Fc receptors can be found for example in U.S. Pat.Nos. 5,624,821 and 5,648,260

Antibody proteins of the presently disclosed and claimed inventiveconcepts can be produced using techniques well known in the art. Forexample, the antibody proteins can be produced recombinantly in cells(79, 86).

For recombinant production, a polynucleotide sequence encoding theantibody protein is inserted into an appropriate expression vehicle,such as a vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence. Theexpression vehicle is then transfected into a suitable target cell whichwill express the peptide. Transfection techniques known in the artinclude, but are not limited to, calcium phosphate precipitation (87)and electroporation (88). A variety of host-expression vector systemsmay be utilized to express the antibody proteins described hereinpreferably including eukaryotic cells.

The presently disclosed and claimed inventive concepts further provideisolated nucleic acids encoding the antibodies disclosed or otherwiseenabled herein. The nucleic acids may comprise DNA or RNA and may bewholly or partially synthetic or recombinant. Reference to a nucleotidesequence as set out herein encompasses a DNA molecule with the specifiedsequence, and encompasses a RNA molecule with the specified sequence inwhich U is substituted for T, unless context requires otherwise.

In another embodiment, the nucleic acid molecules which encode theantibodies of the presently disclosed and claimed inventive conceptsalso comprise nucleotide sequences that are, for example, at least 50%identical to the sequences disclosed herein. Also contemplated areembodiments in which a sequence is at least 85%, 90%, 95%, 96%, 97%,98%, 99%, or 99.5% identical to a sequence disclosed herein and/or whichhybridize to a sequence of the presently disclosed and claimed inventiveconcepts under conditions of high or moderate stringency. The percentidentity may be determined by visual inspection and mathematicalcalculation.

Stringency, including “high stringency,” as used herein, includesconditions readily determined by the skilled artisan based on, forexample, the length of the DNA. Generally, such conditions are definedas hybridization conditions of 50% formamide, 6×SSC at 42° C. (or othersimilar hybridization solution, such as, e.g., Stark's solution, in 50%formamide at 42° C.), and with washing at approximately 68° C., 0.2×SSC,0.1% SDS. The skilled artisan will recognize that the temperature andwash solution salt concentration can be adjusted as necessary accordingto factors such as the length of the probe.

“Moderate stringency,” as used herein, includes conditions that can bereadily determined by those having ordinary skill in the art based on,for example, the length of the DNA. The basic conditions are set forthby Sambrook et al. (79) and include use of a prewashing solution for thenitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization conditions of 50% formamide, 6×SSC at 42° C. (or othersimilar hybridization solution, such as Stark's solution, in 50%formamide at 42° C.), and washing conditions of 60° C., 0.5×SSC, 0.1%SDS.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, generallybeing directed against a single epitopic site. Furthermore, in contrastto conventional polyclonal antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that, in one embodiment, they are synthesized by thehybridoma culture, uncontaminated by other immunoglobulins. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567).

Methods of making antibody fragments are also known in the art (89)(incorporated herein by reference). Antibody fragments of the presentinvention can be prepared by proteolytic hydrolysis of the antibody orby expression in E. coli of DNA encoding the fragment. Antibodyfragments, as noted above, can be obtained by pepsin or papain digestionof whole antibodies conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly. These methods are described, for example,in U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647, and referencescontained therein, which are hereby expressly incorporated in theirentireties by reference.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the conformational epitopethat is recognized by the intact antibody. For example, Fv fragmentscomprise an association of VH and VL chains. This association may benoncovalent or the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde.Preferably, the Fv fragments comprise VH and VL chains connected by apeptide linker. These single-chain antigen binding proteins (sFv) areprepared by constructing a structural gene comprising DNA sequencesencoding the VH and VL domains connected by an oligonucleotide. Thestructural gene is inserted into an expression vector, which issubsequently introduced into a host cell such as E. coli. Therecombinant host cells synthesize a single polypeptide chain with alinker peptide bridging the two V domains. Another form of an antibodyfragment is a peptide coding for a single CDR. CDR peptides (“minimalrecognition units”) are often involved in antigen recognition andbinding. CDR peptides can be obtained by cloning or constructing genesencoding the CDR of an antibody of interest. Such genes are prepared,for example, by using the polymerase chain reaction to synthesize thevariable region from RNA of antibody-producing cells.

The presently disclosed and claimed inventive concepts compriseengineered antibodies including fully human and humanized forms ofnon-human (e.g., primate or murine) antibodies. Such humanizedantibodies are chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) that contain minimalsequences derived from non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins in which residues from aCDR of the recipient are replaced by residues from a CDR of a nonhumanspecies such as mouse, rat or rabbit having the desired specificity,affinity and capacity.

In making an engineered antibody, a DNA sequence encoding an antibodymolecule of the presently disclosed and claimed inventive concepts isprepared synthetically by established standard methods. For example,according to the phosphoamidine method, oligonucleotides aresynthesized, e.g. in an automatic DNA synthesizer, purified, annealed,ligated and cloned in suitable vectors.

The DNA sequence may then be inserted into a recombinant expressionvector, which may be any vector, which may conveniently be subjected torecombinant DNA procedures. The choice of vector will often depend onthe host cell into which it is to be introduced. Thus, the vector may bean autonomously replicating vector, i.e., a vector that exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g., a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

In the vector, the DNA sequence encoding the protein should be operablyconnected to a suitable promoter sequence. The promoter may be any DNAsequence, which shows transcriptional activity in the host cell ofchoice and may be derived from genes encoding proteins either homologousor heterologous to the host cell. Examples of suitable promoters fordirecting the transcription of the coding DNA sequence in mammaliancells include, but are not limited to, the LTR promoter, SV 40 promoter,the MT-1 (metallothionein gene) promoter or the adenovirus 2 major latepromoter. A suitable promoter for use in insect cells is the polyhedrinpromoter. Suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes or alcohol dehydrogenase genes orthe TPI1 or ADH2-4c promoters. Suitable promoters for use in filamentousfungus host cells are, for instance, the ADH3 promoter or the tpiApromoter.

The DNA coding sequence may also be operably connected to a suitableterminator, such as the human growth hormone terminator or (for fungalhosts) the TPI1 or ADH3 promoters. The vector may further compriseelements such as polyadenylation signals (e.g. from SV 40 or titleadenovirus 5 Elb region), transcriptional enhancer sequences (e.g. theSV 40 enhancer) and translational enhancer sequences (e.g., onesencoding adenovirus VA RNAs).

The recombinant expression vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence (when the host cell is a mammalian cell) isthe SV 40 origin of replication. The vector may also comprise aselectable marker, e.g. a gene the product of which complements a defectin the host cell, such as the gene coding for dihydrofolate reductase(DHFR) or one which confers resistance to a drug, e.g. neomycin,hydromycin or methotrexate.

The procedures used to ligate the DNA sequences coding the proteins, thepromoter and the terminator, respectively, and to insert them intosuitable vectors containing the information necessary for replication,are well known to persons skilled in the art.

To obtain recombinant proteins of the presently disclosed and claimedinventive concepts the coding DNA sequences may be usefully fused with asecond peptide coding sequence and a protease cleavage site codingsequence, giving a DNA construct encoding the fusion protein, whereinthe protease cleavage site coding sequence positioned between the HBPfragment and second peptide coding DNA, inserted into a recombinantexpression vector, and expressed in recombinant host cells. In oneembodiment, said second peptide selected from, but not limited by thegroup comprising glutathion-S-reductase, calf thymosin, bacterialthioredoxin or human ubiquitin natural or synthetic variants, orpeptides thereof. In another embodiment, a peptide sequence comprising aprotease cleavage site may be the Factor Xa, with the amino acidsequence IEGR, enterokinase, with the amino acid sequence DDDDK,thrombin, with the amino acid sequence LVPR/GS, or Acharombacterlyticus, with the amino acid sequence XKX, cleavage site.

The host cell into which the expression vector is introduced may be anycell which is capable of expression of the peptides or full-lengthproteins, and is preferably a eukaryotic cell, such as invertebrate(insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes ormammalian cells, in particular insect and mammalian cells. Examples ofsuitable mammalian cell lines include, but are not limited to, theHEk293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC CRL-1632, ATCCCCL-10) or CHO (ATCC CCL-61) cell lines. Methods of transfectingmammalian cells and expressing DNA sequences introduced in the cells arewell known in the art.

Alternatively, fungal cells (including yeast cells) may be used as hostcells. Examples of suitable yeast cells include cells of Saccharomycesspp. or Schizosaccharomyces spp., in particular strains of Saccharomycescerevisiae. Examples of other fungal cells are cells of filamentousfungi, e.g. Aspergillus spp. or Neurospora spp., in particular strainsof Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp.for the expression of proteins is described in, e.g., EP 238 023.

The medium used to culture the cells may be any conventional mediumsuitable for growing mammalian cells, such as a serum-containing orserum-free medium containing appropriate supplements, or a suitablemedium for growing insect, yeast or fungal cells. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes.

The proteins recombinantly produced by the cells may then be recoveredfrom the culture medium by conventional procedures including separatingthe host cells from the medium by centrifugation or filtration,precipitating the proteinaceous components of the supernatant orfiltrate by means of a salt, e.g. ammonium sulphate, purification by avariety of chromatographic procedures, e.g. HPLC, ion exchangechromatography, affinity chromatography, or the like.

The antibodies of the present invention preferably include one or moremodifications which inactivate complement. The term “complementactivity” broadly encompasses the biochemical and physiologicalactivities associated with activation of the complement system,individual complement pathway associated molecules, as well as genesencoding these molecules. Therefore, complement activities include,e.g., structure and expression of a gene encoding a complement pathwaymolecule, biochemical activity (e.g., enzymatic or regulatory) of acomplement pathway molecule, cellular activities that initiate or resultfrom activation of the complement system, and presence of serumautoantibodies against complement pathway molecules. In the hSel001antibody the preferred modification to inactivate complement is areplacement of a lysine residue with alanine at position 342 in theheavy chain constant region CH2. Other substitutions at the sameposition may include for example any of gly, leu, trp, tyr, pro, thr,ser, met, asp, asn, glu, gln, phe, ile, val, thr, and cys with theproviso that the substitution is also effective in eliminating theability of the constant region to activate complement.

The terms “complement pathway associated molecules,” “complement pathwaymolecules,” and “complement pathway associated proteins” are usedinterchangeably and refer to the various molecules that play a role incomplement activation and the downstream cellular activities mediatedby, responsive to, or triggered by the activated complement system. Theyinclude initiators of complement pathways (i.e., molecules that directlyor indirectly triggers the activation of complement system), moleculesthat are produced or play a role during complement activation (e.g.,complement proteins/enzymes such as C3, C5, C5b-9, Factor B, MASP-1, andMASP-2), complement receptors or inhibitors (e.g., clusterin,vitronectin, CR1, or CD59), and molecules regulated or triggered by theactivated complement system (e.g., membrane attack complex-inhibitoryfactor, MACIF). Thus, in addition to complement proteins noted above,complement pathway associated molecules also include, e.g., C3/C5convertase regulators (RCA) such as complement receptor type 1 (alsotermed CR1 or CD35), complement receptor type 2 (also termed CR2 orCD21), membrane cofactor protein (MCP or CD46), and C4bBP; MACregulators such as vitronectin, clusterin (also termed “SP40,40”), CRP,CD59, and homologous restriction factor (HRF); immunoglobulin chainssuch as Ig kappa, Ig lambda, or Ig gamma; C1 inhibitor; and otherproteins such as CR3, CR4 (CD11b/18), and DAF (CD55).

In an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal anti-P-selectin antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with the conformational epitopesdescribed herein respectively to thereby isolate immunoglobulin librarymembers that bind P-selectin in accordance with the present invention.Kits for generating and screening phage display libraries arecommercially available (e.g., the Pharmacia Recombinant Phage AntibodySystem, Catalog No. 27-9400-01; and the Stratagene SurJZAP™. PhageDisplay Kit, Catalog No. 240612).

P-selectin mediates interaction of activated platelets or endothelialcells with blood cells including certain red blood cells (i.e. sickledred cells) and leukocytes including monocytes, neutrophils, eosinophils,CD4⁺T cells, CD8⁺T cells, B cells and natural killer (NK) cells. Asnoted herein, it is known that P-selectin is involved in a number ofcellular responses to inflammation resulting from injury, infection, orphysicochemical assaults. Atherosclerosis, characterized byatherosclerotic lesions on the inner surfaces of blood vessels, is oneexample of a condition involving the binding of certain leukocytes toP-selectin-bearing endothelial cells on the inner lining of blood vesselwalls.

As indicated above, therapies directed to blocking P-selectin function,for example, using antibodies to P-selectin to prevent the tethering androlling of leukocytes and adherence of red blood cells (i.e. sickled redcells), could have a profound effect on numerous types of inflammatoryand thrombotic diseases. Given its pivotal roll in the initiation ofrolling and tethering of leukocytes to the endothelium and platelets,P-selectin is a primary target for therapeutic development to treatinflammatory and thrombotic disorders. For example, the transient natureof the acute phase of sickle cell anemia coupled with the recurrentchronic effects of organ damage and associated complications andmorbidity suggests that a therapeutic intervention that exhibits bothblocking initial adhesion due to binding of P-selectin and PSGL-1, andinducing dissociation of prior, ongoing or preestablished adhesion,would have novel application to this and other inflammatory andthrombotic diseases. The present invention thus encompasses, in oneembodiment, a method of using a conformational antibody binding epitopeof P-selectin to screen for and identify “single function”-blockingantibodies to P-selectin which block P-selectin/PSGL-1 binding, and“dual function” antibodies to P-selectin which not only blockP-selectin-PSGL-1 binding, but which also cause dissociation ofpreformed P-selectin/PSGL-1 complex (and thus cell-cell complex), andthe use of such antibodies for therapeutic treatment of diseases, suchas, but not limited to, inflammatory and thrombotic diseases.

As noted above, novel conformational binding epitopes of P-selectin havebeen discovered. These binding epitopes have enabled the identificationherein of antibodies to human P-selectin which block the binding ofPSGL-1 to P-selectin and thus block the function of P-selectin (thus theantibodies may be referred to herein as “function-blocking” antibodies).The discovery of these conformational epitopes have further led to thediscovery of dual function anti-P-selectin antibodies which bind withhigh specificity to the conformational epitope and which not only blockthe binding of P-selectin and PSGL-1, but which also induce thedissociation of preformed P-selectin/PSGL-1 complexes (i.e., thereversal of P-selectin-PSGL-1 binding) thereby causing the dissociationof cell complexes such as leukocyte/endothelial cell,leukocyte/platelet, lymphocyte/endothelial cell, lymphocyte/platelet,sickled red cell/endothelial cell or sickled red cell/plateletcomplexes.

The binding regions for some antibodies to P-selectin have beenpreviously mapped using constructs of large functional domainsencompassing the lectin, epidermal growth factor (EGF) and consensusrepeat (CR) regions of the native protein P-selectin in mouse and human(90, 70, 91-95). These results indicated the primary binding areas forsome function-blocking antibodies to P-selectin were in the lectinbinding domain, a region that spans amino acid residues 1-120 of thenative P-selectin protein, or in the EGF domain spanning amino acids121-154.

The present disclosure describes the discovery of novel nonlinear (e.g.,conformational) epitopes by using mouse/human chimeras containing thelectin, EGF, CR1 and CR2 domains of P-selectin that were probed withfunction-blocking test antibodies to P-selectin. Previous work has shownthat, at a minimum, expression of the lectin and EGF domains arerequired for proper folding and conformation of P-selectin constructs(91). A comparison of the amino acid sequences of human and mouseP-selectin indicated that there is homology in the lectin domain with aspecific number of amino acid residue differences between human andmouse. Herein, three-dimensional (3-D) homology was used to compare thehuman and mouse lectin, EGF, CR1 and CR2 domains of P-selectin (FIG. 1)to identify amino acid differences between human and mouse P-selectins(sequences and numbering according to the mature proteins).

Where used herein the term “mouse amino acid” refers to an amino acidresidue which is found in mouse P-selectin but is not found in thecorresponding position in human P-selectin. Where used herein the term“human amino acid” refers to an amino acid residue which is found inhuman P-selectin but is not found in the corresponding position in mouseP-selectin.

The method used 3-D modeling of P-selectin to compare the positions ofamino acid differences between human and mouse P-selectin on the exposedsurface of the protein and to identify clusters of amino aciddifferences between human and mouse in the lectin and EGF domains whichare located on the surface of the folded protein. This 3-D methodrepresents clusters of amino acid differences which result fromjuxtaposition of discontinuous amino acids brought into proximity to oneanother by folding of the protein. For example, some amino acids willform conformational epitopes by virtue of being on the same surface,e.g. face, of helical structures. Homology comparison of such clustersallowed for selection of amino acid substitutions to test the effect ofsuch changes on the binding of function-blocking (PSGL-1 blocking)antibodies to human P-selectin.

The method further involved mapping of conserved restriction sites inthe open reading frames of the cDNA to identify a strategy forconstructing chimeric proteins that span the lectin, EGF, CR1 and CR2domains and would enable substitution of single or multiple amino acidsat specific sites in the human or mouse P-selectin to identify thoseamino acids which optimize antibody binding to human P-selectin.Chimeras were constructed with known molecular cloning techniques, usinghuman and mouse P-selectin N-terminal regions spanning the ATG throughCR2 domain with a suitable vector such as pBluescript (pBS-hPsel andpBS-mPsel). The chimeras were inserted into another suitable vector suchas pIG1 (pIG-hPsel and pIG-mPsel) where the constructs were fused to theFc region of human IgG1 containing the hinge, CH2 and CH3 region. Theseconstructs preserved structures that are consistent with the nativeconformation of P-selectin. Thus domains that were exposed on thesurface of the native protein were also present on the chimeraconstructs and thus served as putative epitopes for binding of testantibodies. Such constructs could be transfected and transientlyexpressed using molecular and cell expression techniques known topersons having ordinary skill in the art.

Using this method, test antibodies, can be evaluated for binding to thehuman/mouse chimeras using fluorescence-activated cell sorting (FACS)and surface plasmon resonance (BIACORE) methods known to persons havingordinary skill in the art. The effects of changes in amino acids invarious positions in the chimera constructs by substitution of mouseamino acids, for example, into the human sequence, conversely, or humanamino acids into the mouse sequence, for example, that abrogated orenabled binding of antibodies directed to human P-selectin wereevaluated and thus enabled identification of particular amino acids foroptimal binding.

Characterization of Chimera Constructs

Amino acids of mouse P-selectin which have been substituted into thehuman P-selectin sequence are indicated in boldface in the chimerasdescribed below. Amino acids of human P-selectin which have beensubstituted into the mouse sequence are indicated in italicizedboldface. Substitution of glutamine for histidine is indicated asunderlined boldface.

Native Protein Constructs SEQ ID NO: 1Human P-selectin lectin, EGF, CR1, CR2 domains   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 2Mouse P-selectin lectin, EGF, CR1, CR2 domains-Amino acid differences from human in boldface.  42WTYNYSTKAYSWNNSRVFCRRHFTDLVAIQNKNEIAHLNDVIPFFNSYYWIGIRKINNKW 102TWVGTNKTLTEEAENWADNEPNNKKNNQDCVEIYIKSNSAPGKWNDEPCFKRKRALCYTA 162SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 222FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 282FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIAHuman/Mouse Chimera Constructs SEQ ID NO: 3 Chimera-1(mouse substitutions in human cluster A - N ₄ N ₁₄ V ₁₇ F ₁₈ R ₂₀ R ₂₁ H₂₂ F ₂₃)   1WTYNYSTKAYSWNNSRVFCRRHFTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 4 Chimera-2(human cluster A to I₃₅ - mouse thereafter)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIAHLNDVIPFFNSYYWIGIRKINNKW  61TWVGTNKTLTEEAENWADNEPNNKKNNQDCVEIYIKSNSAPGKWNDEPCFKRKRALCYTA 121SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 241FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA SEQ ID NO: 5 Chimera-3(substitutions in human cluster A - to mouse N ₄ N ₁₄ V ₁₇ F ₁₈)   1WTYNYSTKAYSWNNSRVFCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 6 Chimera-4(substitutions in human cluster A - to mouse R ₂₀ R ₂₁ H ₂₂ F ₂₃)   1WTYHYSTKAYSWNISRKYCRRHFTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 7 Chimera-5(single amino acid change - human H₄ to mouse N ₄)   1WTYNYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDOVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 8 Chimera-5Q(substitution of  Q  for H₄ in cluster A - removes putativeglycosylation site)   1 WTY QYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 9 Chimera-6(human sequence to EGF - S₁₂₁ - mouse EGF, CR1 and CR2)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 241FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA SEQ ID NO: 10 Chimera-7(human sequence to G₁₇₇ - mouse thereafter)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGEFS 181FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 241FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA SEQ ID NO: 11 Chimera-7B(human sequence to end of EGF - V₁₅₆ - mouse thereafter)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 241FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA SEQ ID NO: 12 Chimera-8(single amino acid change - human I₁₄ to mouse N ₁₄)   1WTYNYSTKAYSWNNSRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 13 Chimera-9(single amino acid change - human K₁₇ to mouse V ₁₇)   1WTYHYSTKAYSWNISRVYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 14 Chimera-10(single amino acid change - human Y₁₈ to mouse F ₁₈)   1WTYHYSTKAYSWNISRKFCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 15 Chimera-11(single amino acid change - human Q₂₀ to mouse R ₂₀)   1WTYHYSTKAYSWNISRKYCRNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ 1D NO: 16 Chimera-12(single amino acid change - human N₂₁ to mouse R ₂₁)   1WTYHYSTKAYSWNISRKYCQRRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 17 Chimera-13(single amino acid change - human R₂₂ to mouse H ₂₂)   1WTYHYSTKAYSWNISRKYCQNHYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 18 Chimera-14(single amino acid change - human Y₂₃ to mouse F ₂₃)   1WTYHYSTKAYSWNISRKYCQNRFTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 241FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 19 Chimera-15(human sequence to cluster C2 - S₉₇ - mouse thereafter)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSNSAPGKWNDEPCFKRKRALCYTA 121SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 241FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA SEQ ID NO: 20 Chimera-16(substitution of human 

₄

₁₄

₁₇

₂₁

₂₂ into mouse Cluster A)   1 WTY

YSTKAYSWN

SR

FCR

FTDLVAIQNKNEIAHLNDVIPFFNSYYWIGIRKINNKW  61TWVGTNKTLTEEAENWADNEPNNKKNNQDCVEIYIKSNSAPGKWNDEPCFKRKRALCYTA 121SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 242FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA SEQ ID NO: 21 Chimera-17(human sequence to Cluster B to I₃₅ - mouse Cluster B to I₄₂ -human to CR1 to E₁₅₄ - mouse CR1, CR2)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIAHLNDVIPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 242FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA SEQ ID NO: 22 Chimera-17B(human sequence to Cluster B to I₃₅ - mouse Cluster B to I₄₂-human thereafter)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIAHLNDVIPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 242FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 23 Chimera-18(human sequence with mouse cluster C (C1, C2, C3) and mouse CR1, CR2)  1 WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPFFNSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSNSAPGKWNDEHCLKKKRALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 242FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA SEQ ID NO: 24 Chimera-18B(human sequence with mouse cluster C (C1, C2, C3)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPFFNSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSNSAPGKWNDEHCLKKKRALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 242FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 25 Chimera-19(human sequence with mouse Cluster D and mouse CR1, CR2)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKINNKW  61TWVGTNKTLTEEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 242FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA SEQ ID NO: 26 Chimera-19B(human sequence with mouse Cluster D)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKINNKW  61TWVGTNKTLTEEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 242FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 27 Chimera-20(human sequence with mouse Cluster E and mouse CR1, CR2)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEPCFKRKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVKECGKVNIPQHVLMNCSHPLGEFS 181FNSQCTFSCAEGYELDGPGELQCLASGIWTNNPPKCDAVQCQSLEAPPHGTMACMHPIAA 242FAYDSSCKFECQPGYRARGSNTLHCTGSGQWSEPLPTCEAIA SEQ ID NO: 28 Chimera-20B(human sequence with mouse Cluster E)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEPCFKRKHALCYTA 121SCQDMSCSKQGECLETIGNYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 242FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - - SEQ ID NO: 29 Chimera-21(human sequence with mouse Cluster F)   1WTYHYSTKAYSWNISRKYCQNRYTDLVAIQNKNEIDYLNKVLPYYSSYYWIGIRKNNKTW  61TWVGTKKALTNEAENWADNEPNNKRNNEDCVEIYIKSPSAPGKWNDEHCLKKKHALCYTA 121SCQDMSCSNQGECIETIGSYTCSCYPGFYGPECEYVRECGELELPQHVLMNCSHPLGNFS 181FNSQCSFHCTDGYQVNGPSKLECLASGIWTNKPPQCLAAQCPPLKIPERGNMTCLHSAKA 242FQHQSSCSFSCEEGFALVGPEVVQCTASGVWTAPAPVCK - - -

FACS Analysis of Anti-P-Selectin Antibodies to Human/Mouse Chimeras

Antibody binding to human/mouse chimeras of P-selectin was analyzedusing FACS analysis on a system such as a BD BIOSCIENCES FACS ARIA CELLSORTER to measure binding of anti-P-selectin antibodies to human/mousechimeras which were coupled to beads coated with a goat anti-human Fcantibody. Such beads coated with chimeras were incubated with testanti-P-selectin antibodies that were then detected with anti-mouse Fc orisotype specific antibodies labeled with reporters, such as FITC,suitable for detection by the FACs system.

One-step Surface Plasmon Resonance (BIACORE)

In one aspect of the presently disclosed and claimed inventive concepts,BIACORE chips were used to capture a test anti-P-selectin antibody.Human-mouse P-selectin chimeras described herein were disposed onto thechip and test antibodies were added to the prebound chip. Binding of thechimeras to test antibodies was measured by resonance response units.

Two-Step Surface Plasmon Resonance (BIACORE) Analysis

In another aspect of the presently disclosed and claimed inventiveconcepts, a capture chip, such as a BIACORE chip was provided with agoat anti-human IgG Fc polyclonal antibody covalently attached to itssurface. P-selectin chimeric human/mouse constructs of the lectin, EGF,CR1 and CR2 domain on a human IgG Fc were injected onto the chip andcaptured at concentrations that achieve a standardized level of surfacecoating as measured by the resonance response. The resonance responselevel achieved after loading each P-selectin chimera construct wasdesignated as a new “secondary baseline” level. Test anti-P-selectinantibodies (e.g., mouse monoclonal anti-P-selectin antibodies G1, G3 andG5) were then injected onto the BIACORE chip and incubated for bindingto the P-selectin chimera construct already captured on the surface. Themethod could be modified to test humanized antibodies by creatingP-selectin constructs on mouse IgG Fc and capturing with a goatanti-mouse IgG Fc polyclonal antibody and then probing with testhumanized anti-P-selectin antibodies. Antibodies which bind to theP-selectin constructs cause an increase of the resonance response levelfrom the secondary baseline. The resulting increase in resonanceresponse may be measured as “added resonance units (RUs)” and thusindicate the level of binding to the P-selectin construct pre-coatedonto the capture chip of the test antibody. Using these methods, optimalrequirements for the binding of anti-P-selectin antibodies to P-selectinchimeras were precisely mapped to particular conformational epitopes.

Identification of Dual Function Anti-P-Selectin Antibodies (Antibodiesthat Both Block and Dissociate Binding of P-Selectin to PSGL-1)

BIACORE analysis was also used to discover dual functionality ofspecific anti-P-selectin antibodies, i.e., as discussed above, they canboth block and dissociate (reverse) binding interactions betweenP-selectin and PSGL-1. In this method, PSGL-1, or small moleculemimetics of the binding epitope of PSGL-1 such as a biotinylatedglycosulfopeptide mimetic (e.g., GSP-6), or chimeric proteins containingthe N-terminus of PSGL-1, are captured on a BIACORE chip, such as astreptavidin chip, using methods known to persons having ordinary skillin the art (GSP-6 is a glycosylated, sulfated 18 amino acid peptidemimetic of amino acids 42-60 of the exposed N-terminus of PSGL-1described in detail in U.S. Pat. No. 6,593,459, for example). First, todemonstrate “function-blocking” ability, in one embodiment, ananti-P-selectin antibody is pre-mixed with soluble P-selectin andincubated for a period to allow formation of the P-selectin/antibodycomplex. The resulting anti-P-selectin antibody/P-selectin complex isintroduced onto the chip bearing the PSGL-1 (or PSGL-1 mimetic) andbinding to the PSGL-1 or its mimetic is measured. Anti-P-selectinantibodies, which prevent binding of P-selectin to the PSGL-1 or PSGL-1mimetic on the chip, are designated as function-blocking antibodies.

Second, anti-P-selectin antibodies which have been shown (by theabove-method or another similar method) to be function-blockingantibodies (i.e., which block PSGL-1 binding to P-selectin), can betested for an additional function, that is, having the ability todissociate (reverse) binding between preformed P-selectin PSGL-1complex. Such antibodies can be tested for “dual function” propertiesusing the method of BIACORE analysis discussed herein. In oneembodiment, to demonstrate the dual function property, PSGL-1, or amimetic thereof such as GSP-6, is coupled to a BIACORE chip. P-selectinis then disposed on the chip and allowed to bind to the PSGL-1, or themimetic. After equilibrium binding of P-selectin to PSGL-1, or themimetic, is indicated by the sensogram, function-blockinganti-P-selectin antibodies are introduced and the dissociation ofP-selectin binding to PSGL-1, or the mimetic is measured by anyappropriate method. Such antibodies that are shown to dissociate (i.e.,reverse), P-selectin/PSGL-1 binding are designated as “dissociatingantibodies” and are characterized as dual function antibodies, i.e.,they possess both function-blocking and dissociating properties indisrupting binding of P-selectin to PSGL-1. Such dual functionantibodies are a particularly preferred embodiment of the invention asthey are especially suitable for therapeutic application as treatmentsof acute and chronic inflammatory and thrombotic diseases such as aredescribed elsewhere herein.

Discovery of Conformational Epitopes

The three-dimensional (3-D) structure of the mature human and mouseP-selectin proteins were analyzed and compared as to amino aciddifferences in the lectin and EGF domains. Six clusters ofconformational amino acid differences were identified on exposedsurfaces of the proteins. These were designated as clusters A, B, C, D,E and F (FIG. 1). The N-termini of human and mouse P-selectins spanningresidues 1-35 contain 8 amino acid differences. Cluster A wasarbitrarily defined by the boundary of the first amino acid difference(H₄) and the last amino acid difference (Y₂₃). Cluster A contains 20amino acids and forms a rigid alpha helix with a cysteine bond near theN-terminus of the protein (see region “1” in FIG. 5). Cluster B (FIG. 1)is a conformational epitope spanning amino acid residues 36-42 andcontains 4 amino acid differences between human and mouse P-selectin.Where used herein, the term “conformational epitope” is intended torefer to an epitope which is not recognized under reducing conditions.Clusters C and E (FIG. 1) are conformational and discontinuous and arebrought into proximity by folding of the native P-selectin protein.Cluster C has three conformational regions (C1, C2, C3) containing 5amino acid differences between human and mouse P-selectin. Cluster C1 isseparated from C2 by 51 amino acids and cluster C2 is separated from C3by 15 amino acids. Likewise cluster E has two conformational epitopes(E1, E2) containing five amino acid differences between human and mouseP-selectin with cluster E1 being separated from E2 by 19 amino acids.Clusters A, B, C, D and E lie within the consensus lectin domain ofP-selectin (FIG. 1). Cluster F resides in the EGF domain and has 3 aminoacid differences out of 11 amino acids. Clusters C1, E1, C2, E2 and C3encompass key contact residues which have previously been identified forinteraction of P-selectin with its physiological ligand PSGL-1 (Somerset al). Clusters A and B are distal to (upstream of) these contactresidues.

The open reading frames of cDNAs for human and mouse P-selectin wereanalyzed to identify common restriction sites that could be used toassemble chimeras spanning the clusters. PCR and chemical DNA synthesiswas used to generate cDNAs coding for specific protein or chimeraconstructs such as SEQ ID NO: 1-29 (described above, and in the SequenceListing). Restriction cloning was used to construct plasmids coding forthe human/mouse chimeras. The chimeras were transiently expressed inCOS-7 cells and utilized for FACs and BIACORE analysis. P-selectinchimeras were tested for binding function using BIACORE by analyzingtheir binding to GSP-6 bound to a BIACORE chip. As noted above, GSP-6 isa small molecule that mimics the N-terminus of human PSGL-1 (96). Alltested chimeras bound to the GSP-6 on the chip, though to varyingdegrees, as mouse P-selectin has a lower binding affinity to humanPSGL-1 than does human P-selectin. This indicated that chimeras hadmaintained function after expression and purification.

FACS Analysis

The results of FACs analysis of anti-P-selectin antibodies, using theconstructs or chimeras corresponding to SEQ ID NOs.:1-29 are summarizedin Table 1 (below). Three anti-P-selectin test antibodies (G1, G3 andG5) were isolated from hybridomas generated by immunization of mice witha human recombinant P-selectin containing the lectin and EGF domains(90). Previous studies had shown that these antibodies were specific tohuman P-selectin and that G1 and G3 are function-blocking antibodies andG5 is non-blocking (90, and unpublished data). G1 and G3 were determinedherein to bind to human P-selectin (SEQ ID NO:1) but did not bind tomouse P-selectin (SEQ ID NO:2). When the corresponding eight mouse aminoacids were substituted in cluster A of the human sequence (Chimera 1,SEQ ID NO:3), binding by G1 antibody was abolished, indicating that atleast one or more of the corresponding eight amino acid positions incluster A was essential for binding of the G1 antibody to P-selectin andthat the substitution with the “mouse” amino acids in those one or morepositions abolished the binding. To further evaluate the bindingspecificity of the G1 test antibody, the eight different human aminoacids in positions 1-23 were substituted in cluster A of the mousesequence (SEQ ID NO:4, chimera-2) and G1 binding was achieved. Chimerascontaining the human P-selectin lectin domain with mouse amino acidsubstitutions in the EGF, CR1 and CR2 domains (SEQ ID NO:9, chimera-6),and human sequence through the EGF domain with mouse CR1 and CR2 domains(SEQ ID NO:11, chimera-7B), were bound by G1 and G3 indicating that theprimary binding epitopes remained intact after substitution of thesemouse amino acids and that the conformation of the antibody bindingepitopes were not adversely affected.

Using these methods, the test antibody G3 was shown to bind humanP-selectin, did not bind mouse P-selectin and in contrast to G1, boundto SEQ ID NO:3 (chimera-1), indicating that G3 binds to an epitopedistinct from the epitope bound by G1. Specifics of the G3 epitopemapping are outlined in Table 1 below.

The test antibody G5, previously shown to be non-blocking, was alsoanalyzed using this method. G5 was shown to be specific for humanP-selectin, did not bind mouse P-selectin, and was confirmed as anon-blocking antibody. G5 bound to SEQ ID NO:1 (human P-selectin), SEQID NO:3, and SEQ ID NO:10 that spans the EGF domain and includes thefirst part of CR1 to N₁₇₈, but G5 did not bind to SEQ ID NO:2 (mouseP-selectin) or to SEQ ID NO:9 that spans to S₁₂₈, or to SEQ ID NO:11that spans to the start of CR1 at V₁₅₆. These results indicate thatantibody G5 binds to the first part of CR1 and requires at least aminoacids R₁₅₇ through N₁₇₈.

TABLE 1 Results of binding of various antibodies (G1, G3, G4, G5,hSel001) to human and mouse P-selectin and chimera constructs thereof.Human mouse Chimeras Mouse Domains Inserted SeqId in Human P-selectinHuman Mouse G1 G3 G5 G4 hSel001 1 none  1-279  0 +^(1, 2, 3) +^(1, 2, 3)+^(1, 3) +³ +² 2 all  0  1-282 −^(1, 2, 3) −^(1, 2, 3) −^(1, 3) −³ −² 3A_(4, 14, 17, 18, 20, 21, 22, 23) 1-3, 24-279  4-23 −^(1, 2, 3) +^(1, 3)+¹ −³ 4 B, C, D E, F  1-35  36-282 +^(1, 2, 3) −³ +³ 5 A_(4, 14, 17, 18) 19-279  1-18 −² 6 A₂₀₋₂₃ 1-19, 24-279 20-23 −² 7 A₄ 1-3, 5-279  4+^(2w, 3w) +³ +^(3w) 8 none 1-3, 5-279  0 +^(2, 3) +³ +³ 9 F, CR1, CR2 1-128 129-282 +^(1, 2, 3) +^(1, 3) −^(1, 3) +³ 10 CR1, CR2  1-177178-282 +^(1, 2, 3) +^(1, 3) +^(1, 3) +³ 11 CR1, CR2  1-156 157-282 +²+² −² 12 A₁₄ 1-13, 15-279 14 −² 13 A₁₇ 1-16, 18-279 17 −² 14 A₁₈ 1-17,19-279 18 +² 15 A₂₀ 1-19, 21-279 20 +² 16 A₂₁ 1-20, 22-279 21 +^(2w) 17A₂₂ 1-21, 23-279 22 −² 18 A₂₃ 1-22, 24-270 23 +² 19 C2, E2, C3, F, CR1,CR2  1-97  98-282 +^(2, 3) −³ +³ 20 B, C, D E, F 1-35 H/M hybrid  36-282+^(2, 3) −^(2, 3) +³ +² 21 B, CR1, CR2 1-35, 43-156 36-42, 157-282 +³ +³−³ 22 B 1-35, 43-279 36-42 +³ +³ 23 C1, C2, C3, CR1, CR2 1-43, 47-97,99-113, 44-46, 98, 114, 157-282 +³ −³ −³ 115-156 24 C1, C2, C3 1-43,47-97, 99-113, 44-46, 98, 114 −³ +³ 115-279 25 D, CR1, CR2 1-55, 72-15656-71, 157-282 +³ +³ 26 D 1-55, 72-279 56-71 +³ +³ 27 E1, E2, CR1, CR21-84, 89-107, 113- 85-88, 108-112, 157-282 +³ +³ 156 28 E1, E2 1-84,89-107, 113- 85-88, 108-112 +³ +³ 279 29 F 1-128, 140-279 129-139 +³ +³Critical Amino Acid A (4, 14, 17, C First part of A(4, 14, A (4, 14, 17,Postions: 21, 22) CR1 (157- 17, 21, 22) 21, 22) 164) ¹By FACS, ²ByBIACORE 1-step, ³By BIACORE 2-step, ^(W)Weak binding

One-step Surface Plasmon Resonance (BIACORE)

To further investigate the importance of the cluster A domain (aminoacids 4-23) to the binding of G1 antibody to P-selectin, severalchimeric constructs were made in which single or multiple mouse aminoacids were inserted into the human P-selectin sequence and binding to G1was analyzed using the surface plasmon resonance (“one-step” BIACORE)methods disclosed herein. The one-step BIACORE binding results arepresented in Table 1. The G1 test antibody was captured on a BIACOREchip and the binding of various chimeras was measured as response units.A representative sensogram showing binding of G1 to constructs of humanP-selectin (SEQ ID NO:1), mouse P-selectin with human cluster A (SEQ IDNO:4) and mouse P-selectin (SEQ ID NO:2) is shown in FIG. 2. Using thismethod, it was shown that G1 bound to human P-selectin and to SEQ IDNO:4 (where human amino acids were substituted in cluster A of mouseP-selectin), but did not bind to mouse P-selectin (SEQ ID NO:2), whichshowed this method to be consistent with previous results.

Mouse P-selectin (SEQ ID NO:2) has a putative glycosylation site (N) atposition 4 whereas human P-selectin (SEQ ID NO:1) does not. To test theimportance of this difference, SEQ ID NO:1 was substituted at position 4with N (forming SEQ ID NO:7) and Q (forming SEQ ID NO:8) were made inthe human chimera and its effect on G1 antibody binding measured.Inserting N into human P-selectin at position 4 diminished G1 bindingsuggesting that glycosylation at this site in human P-selectin wouldinterfere with antibody binding. Substitution of glutamine (Q) at thisposition did not prevent G1 binding.

To further identify amino positions in cluster A that are optimal oressential for G1 antibody binding, single amino acid substitutions ofmouse sequence amino acids into the human P-selectin (SEQ ID NO:1) weremade, and binding of the resulting chimeras to G1 was measured using theone-step BIACORE method disclosed herein. The chimeras tested (andsubstitution) were: SEQ ID NO:7 (H₄N); SEQ ID NO:12 (I₁₄N); SEQ ID NO:13(K₁₇V); SEQ ID NO:14 (Y₁₈F); SEQ ID NO:15 (Q₂₀R); SEQ ID NO:16 (N₂₁R);SEQ ID NO:17 (R₂₂H); and SEQ ID NO:18 (Y₂₃F). The G1 antibody bound toSEQ ID NO:14, 15 and 18, but did not bind SEQ ID NO:12, 13, and 17 andonly weakly to SEQ ID NO:7 and SEQ ID NO:16. This result confirmed theresults, shown with SEQ ID NO:20, that amino acid positions 4, 14, 17,21, and 22 are each individually required positions for G1 binding. In apreferred embodiment, these amino acids are H₄, I₁₄, K₁₇, N₂₁ and R₂₂,respectively.

Two-step Surface Plasmon Resonance (BIACORE)

To assess G1 binding to additional chimeras and the binding of otheranti-P-selectin test antibodies including G3 and G5 (non-blocking), thetwo-step surface plasmon resonance (“two-step” BIACORE) assay describedherein was used. The results of the “two-step” assays for testantibodies G1, G3 and G5 are presented in Table 1, and in FIG. 3. Usingthis method, none of the test antibodies investigated bound to mouseP-selectin and all bound to human P-selectin demonstrating theirspecificity to human P-selectin. G1, G3 and G5 test antibodies all boundto SEQ ID NO:11 indicating they all bind to a region spanning theN-terminus through the lectin and EGF domains of human P-selectin. TheG5 non-blocking antibody did not bind to SEQ ID NO:9, but did bind SEQID NO:10 confirming that G5 binds to the CR1 domain. G5 bindingproperties were not further evaluated.

Further analysis using this method showed that G3 did not bind SEQ IDNO:23, which has mouse amino acids inserted in cluster C1, C2, C3, CR1,and CR2, nor did it bind SEQ ID NO:24 which has mouse amino acids in C1,C2, and C3. G3 also did not bind to other chimeras that had mousesequence in cluster C, that is SEQ ID NO:19 and SEQ ID NO:20. Theseresults indicate that the blocking test antibody G3 requires cluster Cfor binding and demonstrates the novel finding that conformationalclusters of amino acids brought into proximity by protein folding canserve as binding domains (conformational epitopes) for anti-P-selectinantibodies. The method also confirmed that G3 can block binding ofPSGL-1 and P-selectin and thus has function-blocking properties (FIG.4). However, the method also showed that G3 did not bind to ordissociate (reverse) the binding of P-selectin/PSGL-1 complex (FIG. 4)and thus does not have the dual function properties of the preferredantibodies of the presently disclosed and claimed inventive concepts.

Amino acid positions which optimize binding of G1 were identified bygenerating a human/mouse hybrid in cluster A. The hybrid cluster Achimera (SEQ ID NO:20) contains human P-selectin amino acids atpositions 4, 14, 17, 21 and 22 (H, I, K, N, and R, respectively) andmouse P-selectin amino acids at positions 18, 20, and 23 (Y, Q, and Y,respectively). As indicated in Table 1, G1 bound SEQ ID NO:20. Thisresult when taken with the previous data indicates that amino acidpositions 4, 14, 17, 21, and 22 comprise positions which are eachrequired for optimal binding to P-selectin. These results comprise anovel finding that G1 binds an epitope in the helix structure of clusterA that is distal to the lectin-ligand binding domain contact residuespreviously identified for P-selectin (71). In a preferred embodiment theamino acids at positions 4, 14, 17, 21, and 22 are H, I, K, N, and R,respectively. 3-D analysis of this epitope revealed a rigid helicalstructure with the required amino acids occupying sites on the same faceof the helix; thus cluster A is designated as comprising aconformational epitope (FIG. 5). BIACORE analysis shown in FIG. 4confirmed that G1 can block and also dissociate the binding ofP-selectin and PSGL-1 and thus this antibody has the dual functionproperties of the preferred embodiments of the presently disclosed andclaimed inventive concepts.

These results indicate that antibodies that bind to a conformationalepitope located within amino acids 1-35, and more particularly withinamino acids 4-23, of SEQ ID NO:1 which is distal to the lectin-ligandbinding domain in human P-selectin, will have unique dual functionproperties. Without wanting to be bound by theory, it is contemplatedthat the antibodies which bind to this epitope act by contributingallosteric forces that exert on the lectin-ligand binding interface toinduce a conformational change that dissociates P-selectin binding toPSGL-1. Thus G1 is able to bind the distal epitope at cluster A andblock and dissociate the complex by binding and disrupting the molecularinteractions at the lectin-ligand binding site on P-selectin. Incontrast, antibodies such as G3, that bind to an epitope in thelectin-ligand binding domain of P-selectin can block P-selectin bindingto PSGL-1 by allosteric hindrance, but cannot cause dissociation of theP-selectin/PSGL-1 complex since the antibody cannot bind to theconformational epitope of Cluster C when it is occupied by the ligand.The test antibody G5 bound to the cluster of P-selectin CR1 and wasshown to be non-blocking (FIG. 4).

In summary, antibodies which bind to P-selectin have been characterizedas having three possible activities in regard to the interaction ofP-selectin, and its ligand, PSGL-1. First, P-selectin antibodies canbind to P-selectin but not interfere with the binding of PSGL-1 toP-selectin (“non-blocking”). For example, as shown herein, thenon-blocking antibody G5 binds amino acids 157-164 and requires R₁₅₇,E₁₆₁, L₁₆₂, E₁₆₃ and L₁₆₄ of CR1 for binding. Second, P-selectinantibodies can bind to P-selectin and interfere with the binding ofPSGL-1 to P-selectin (“function-blocking”), but not interfere with apreformed P-selectin/PSGL-1 complex. For example, the results describedherein also showed that antibody G3 binds to conformational clusters ina different part of P-selectin that span the lectin-ligand bindingdomain. G3 binds cluster C and requires C1 amino acids Y₄₄, Y₄₅, S₄₆ andC2 amino acid P₉₈ and C3 amino acid H₁₁₄ and thus requires aconformational epitope. Third, P-selectin antibodies can bind toP-selectin and block the binding of PSGL-1 to P-selectin(function-blocking antibody) and furthermore can cause reversal ofpreformed P-selectin/PSGL-1 binding (dissociative binding). Suchantibodies are referred to herein as “dual function” antibodies. Theresults disclosed herein demonstrate, for example, that the testantibody G1 binds a conformational epitope in cluster A, and that G1binding had an absolute requirement for a conformational epitopecomprising amino acid positions 4, 14, 17, 21, and 22, preferablywherein those amino acids are H, I, K, N, and R, respectively. Asdiscussed elsewhere herein, substitution of the “human” amino acid (H,I, K, N, and R) at any one of these positions, respectively, with thecorresponding “mouse” amino acid (N,N, V, R, and H) will result in theabrogation of binding by the dual function antibodies described andclaimed herein.

In another embodiment of the presently disclosed and claimed inventiveconcepts, a previously uncharacterized mouse monoclonal anti-P-selectinantibody clone designated G4, generated using standard hybridomamethods, was tested for binding to the conformational epitope of clusterA, and was tested for dual function capabilities using the methodsdescribed herein. G4 was tested for binding to human/mouse chimeras SEQID NOs.:1-4, 7-10, 19 and 20. G4 was shown to bind SEQ ID NO:20 and hadsimilar binding specificity as described for G1 (see Table 1 and FIG.3). G4 was then shown to block binding of P-selectin to PSGL-1 (FIG. 4)and also to cause dissociation of preformed P-selectin/PSGL-1 complex,thus characterizing G4 as a dual function P-selectin antibody whichbinds an epitope (in cluster A) which is distal to the lectin-ligandbinding domain of P-selectin and blocks and dissociates binding ofP-selectin to PSGL-1. The G1 and G4 antibodies thus both bound to anepitope in cluster A and both demonstrated dual function properties.This result demonstrates the use of cluster A or specific bindingpositions or amino acids thereof as an epitope able to be used to screenanti-P-selectin antibodies for dual function capabilities (as well asfor function-blocking activity alone). Such dual function antibodiespossess unique properties for therapeutic applications where initiationof P-selectin-mediated adhesion and/or ongoing P-selectin-mediatedadhesion in acute or chronic settings may be treated. Using the methodsdescribed herein, other antibodies having dual function properties (aswell as for function-blocking activity alone) can be identified usingthe method of screening using the cluster A epitope or specificpositions or amino acids thereof.

In another embodiment of the presently disclosed and claimed inventiveconcepts, a humanized IgG2 anti-P-selectin antibody lacking effectorfunction called hSel001 (a humanized P-selectin binding antibodycomprising CDRs of mouse antibody G1 grafted into human frameworkregions and previously characterized in U.S. patent application Ser. No.12/516,987) was also screened using the screening method describedherein. A summary of the data (Table 1) shows that hSel001 antibodybound to the same chimeras (SEQ ID NO:4, 7, 8, 9, 10, 19 and 20) asantibody G1. hSel001 binding was specific to the conformational epitopedescribed herein located within cluster A. Results showed that antibodyhSel001 possesses dual function properties enabling it to both blockbinding of P-selectin to PSGL-1 and dissociate preformedP-selectin/PSGL-1 complexes (FIG. 4). Thus hSel001 is another antibodyencompassed by the presently disclosed and claimed inventive conceptsand can be used as a therapeutic treatment for inflammatory andthrombotic diseases as described herein, and wherein P-selectin bindingto PSGL-1 is blocked, and dissociation of preformed P-selectin/PSGL-1complex is promoted.

Cell-Based In Vitro Rolling Assays Under Flow with Human Neutrophils

To further evaluate the blocking and dissociative properties ofantibodies G1, G3 and hSel001, cell-based in vitro rolling assays wereperformed with freshly isolated human neutrophils that were introducedunder a flow of 1.0 dyn/cm² in a flow chamber coated with low and highlevels of membrane P-selectin. The low density P-selectin was coated at0.25 ug/ml and the high density P-selectin was coated at 2 ug/ml. Sitedensities were determined using I¹²⁵-labeled G1 mAb to be 50 sites/mm²(low) and 380 sites/mm² (high). On low density P-selectin, neutrophilsrolled at an average velocity of 5 μm/s. On high density P-selectin,neutrophils rolled at an average velocity of 1 μm/s. Neutrophils areintroduced in buffer under flow and allowed to begin rolling andtethering. Once equilibrated, test antibodies (G1, G3, hSel001) wereintroduced in cell-free buffer under flow. There is a dead volume ofabout 1 minute interval before the antibody reaches the chamber. At 1minute intervals thereafter, cells remaining bound are counted andexpressed as % cells bound. Results were recorded on video microscopyfor approximately 0-20 minutes and the data analyzed post run.

Results in FIG. 6 panel (A) showed that neutrophils rolled at a highervelocity on low density P-selectin. Thus as the P-selectin/PSGL-1complex released due to normal on/off kinetics of the lectin/ligandbinding, neutrophils traveled greater distances at higher velocity tothe next P-selectin binding site. As the complex releases, thepreviously occupied P-selectin becomes available for binding byanti-P-selectin antibodies. Thus all three antibodies, G1, G3, andhSel01, showed equivalent blocking functionality over the course of 1-4minutes.

Results in FIG. 6 panel (B) on high density P-selectin showed thatneutrophils roll much slower (1 μm/s) as a greater number of P-selectinbinding sites are available. Many neutrophils come to a rolling stop onP-selectin at this density. Under these conditions the murine antibodyG1 and the humanized antibody hSel001 were able to release rolling andtethering neutrophils by dissociating the P-selectin/PSGL-1 compleximmediately and over the course of 1-8 minutes. In contrast the G3anti-P-selectin antibody required up to 20 minutes to block rollingneutrophils. This indicated that the G3 antibody was only able to bindunoccupied P-selectin sites and thus block P-selectin/PSGL-1 complexes,but was not able to bind and dissociate the pre-formed complex. Thesecell-binding assays under flow confirm the BIACORE results reportedpreviously herein which demonstrate that murine antibody G1 andhumanized antibody hSel001 both have dual function properties causingboth blockage and dissociation of preformed P-selectin/PSGL-1 complexes.Thus the hSel001 antibody has the dual function anti-P-selectinproperties of the preferred embodiments of the presently disclosed andclaimed inventive concepts.

The isolated dual function antibody claimed as an embodiment of thepresently disclosed and claimed inventive concepts does not comprise themurine antibody G1.

Antibodies of the presently disclosed and claimed inventive conceptsprovided by any of the above described methods are preferably used inthe manufacture of a pharmaceutical composition for the therapeutictreatment of a pathological condition, wherein such treatment comprisesmitigating, reversion, or inhibiting an inflammatory response orthrombosis.

It is an important objective of the presently disclosed and claimedinventive concepts to use the antibodies, or functionally activefragments or variants of said antibodies for the manufacture of apharmaceutical composition for prevention and/or treatment ofinflammatory responses or diseases or thrombosis such as describedherein.

The presently disclosed and claimed inventive concepts in particular aredirected to using such single and dual function anti-P-selectinantibodies or antibody fragments as described and identified herein intreatments for inflammatory, thrombotic or other conditions or disordersin primates (including humans) which involve platelet, sickled red cell,leukocyte, lymphocyte, and/or endothelial cell adhesion, wherein thecondition or disorder comprises or is associated with (but not limitedto) at least one of sickle cell vasoocclusive pain crisis, inflammatorybowel disease (e.g., Crohn's Disease, ulcerative colitis, enteritis),arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriaticarthritis), graft rejection, graft versus host disease, asthma, chronicobstructive pulmonary disease, psoriasis, dermatitis, sepsis, nephritis,lupus erythematosis, scleroderma, rhinitis, anaphylaxis, diabetes,multiple sclerosis, atherosclerosis, thrombosis, tumor metastasis,allergic reactions, thyroiditis, ischemic reperfusion injury (e.g., dueto myocardial infarction, stroke, or organ transplantation), andconditions associated with extensive trauma, or chronic inflammation,such as for example, type IV delayed hypersensitivity, associated forexample with infection by Tubercle bacilli, or systematic inflammatoryresponse syndrome, or multiple organ failure. The term “primate” as usedherein refers to humans, monkeys, including baboons and cynomolgusmonkeys, and apes, the latter including chimpanzees, gorillas, gibbonsand orangutans, for example.

In the pharmaceutical composition of a medicament according to thepresently disclosed and claimed inventive concepts, the antibodies maybe formulated by any of the established methods of formulatingpharmaceutical compositions, e.g. as described in the latest edition ofRemington's Pharmaceutical Sciences or described elsewhere herein. Thecomposition may typically be in a form suited for local or systemicinjection or infusion and may, as such, be formulated with sterile wateror an isotonic saline or glucose solution. The compositions may besterilized by conventional sterilization techniques, which are wellknown in the art. The resulting aqueous solutions may be packaged foruse or filtered under aseptic conditions and lyophilized, thelyophilized preparation being combined with the sterile aqueous solutionprior to administration. The composition may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as buffering agents, tonicity adjusting agents and thelike, for instance sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, etc. The concentration of proteinsmay vary widely, for example, from less than about 0.01% to as much as15-20% or more by weight. A unit dosage of the composition may containfor example from about 1 μg to about 1000 mg of an antibody or antibodyfragment.

The antibodies or antibody fragments of the presently disclosed andclaimed inventive concepts may be administered topically or byinjection. Dosages will be prescribed by the physician according to theparticular condition and the particular individual to be treated.Dosages and frequency is carefully adapted and adjusted according toparameters determined by the physician in charge. Preferredadministration routes may be oral, via inhalation, subcutaneous,intravenous, intramuscular, intratracheal, intravesical, orintraperitoneal injections and may be given per 24 to 48 hours, or perweek, every 14 days, every 4 weeks for example in the range of from0.01-1000 mg, especially 0.1 mg to 100 mg, in particular 1-10 mg per kgbody weight. The dose may be administered continuously through acatheter or in individual boluses. The antibody of the invention may beadministered in an efficacious quantity such as, but not limited to, theranges between 1 ng/kg to 1 μg/kg, 0.01 μg/kg to 50 μg/kg, 0.1 μg/kg to1 μg/kg, 1 μg/kg to 5 μg/kg, 5 μg/kg to 10 μg/kg, 10 μg/kg to 50 μg/kg,50 μg/kg to 100 μg/kg, 100 mg/kg to 1 mg/kg, 1 mg/kg to 10 mg/kg, or 10mg/kg to 100 mg/kg body weight.

Pharmaceutical compositions used in the presently disclosed and claimedinventive concepts comprising antibodies described herein mayadditionally be supplemented by other therapeutic compounds which areroutinely prescribed by the physician according to the particularcondition and the particular individual to be treated such as ananti-inflammatory drug, wherein said drugs are prescribed by thephysician according to the particular condition and the particularindividual to be treated.

As noted elsewhere herein, the phenomenon of P-selectin/PSGL-1 bindinghas functional importance in sickled red cell, endothelial cellleukocyte and platelet interactions, and/or microvesicle adhesion,leukocyte rolling, recruitment, aggregation; leukocyte secretion ofcytokines; promotion of coagulation; and other aspects of inflammation,thrombosis, coagulation, immune response, and signal transductionincluding, but not limited to, sickle cell vasoocclusive pain crisis,inflammatory bowel disease (e.g., Crohn's Disease, ulcerative colitis,enteritis), arthritis (e.g., rheumatoid arthritis, osteoarthritis,psoriatic arthritis), graft rejection, graft versus host disease,asthma, chronic obstructive pulmonary disease, psoriasis, dermatitis,sepsis, nephritis, lupus erythematosis, scleroderma, rhinitis,anaphylaxis, diabetes, multiple sclerosis, atherosclerosis, thrombosis,tumor metastasis, allergic reactions, thyroiditis, ischemic reperfusioninjury (e.g., due to myocardial infarction, stroke, or organtransplantation), and conditions associated with extensive trauma, orchronic inflammation, such as for example, type IV delayedhypersensitivity, associated for example with infection by Tuberclebacilli, or systematic inflammatory response syndrome, or multiple organfailure. A neutralizing antibody to P-selectin as described herein willinhibit one or more of these activities in a patient as mediated throughP-selectin/PSGL-1 receptor binding (or in the case of sickled red cells,P-selectin/PSGL-1 like receptor binding), in vivo or in vitro, forexample. Thus, the inhibition of P-selectin/PSGL-1 binding with aneutralizing antibody described herein is useful in the treatment in apatient of various conditions and disorders including but not limitedto, those described herein.

The P-selectin specific antibodies or binding fragments described hereincan be linked to another molecule. For example, antibodies may be linkedto another peptide or protein, toxin, radioisotope, cytotoxic orcytostatic agents. The antibodies can be linked covalently by chemicalcross-linking or by recombinant methods. The antibodies may also belinked to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192; or 4,179,337. The antibodies can be chemicallymodified by covalent conjugation to a polymer, for example, to increasetheir stability or half-life. Exemplary polymers and methods to attachthem are also shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285;and 4,609,546.

The antibodies may also be tagged with a detectable label. A detectablelabel is a molecule which, by its chemical nature, provides ananalytically identifiable signal which allows the detection of amolecular interaction. A protein, including an antibody, has adetectable label if it is covalently or non-covalently bound to amolecule that can be detected directly (e.g., by means of a chromophore,fluorophore, or radioisotope) or indirectly (e.g., by means ofcatalyzing a reaction producing a colored, luminescent, or fluorescentproduct). Detectable labels include a radiolabel such as 131I or 99Tc, aheavy metal, or a fluorescent substrate, such as Europium, for example,which may also be attached to antibodies using conventional chemistry.Detectable labels also include enzyme labels such as horseradishperoxidase or alkaline phosphatase. Detectable labels further includechemical moieties such as biotin, which may be detected via binding to aspecific cognate detectable moiety, e.g., labeled avidin.

The presently disclosed and claimed inventive concepts are also directedto methods of screening for anti-P-selectin antibodies and bindingfragments thereof which block both P-selectin/PSGL-1 binding and/orwhich cause dissociation of preformed P-selectin/PSGL-1 complexes.

As noted above, the presently disclosed and claimed inventive conceptsare directed to antibodies against P-selectin, host cells that producesuch anti-P-selectin antibodies, vectors that contain DNA which encodesuch anti-P-selectin antibody expression and screening methods toidentify anti-P-selectin antibodies which block P-selectin/PSGL-1binding and in a further embodiment has a “dual function” in alsocausing dissociation of preformed P-selectin/PSGL-1 complex. Thus, inone embodiment the presently disclosed and claimed inventive concepts isdirected to methods of identifying anti-P-selectin antibodies thatspecifically bind to a conformational epitope in amino acids 1-35, andmore preferably in amino acids 4-23, of human P-selectin (SEQ ID NO:1)(such as the conformational epitope described herein) and which blockPSGL-1, or mimetics thereof, from binding to P-selectin, and which canreverse such binding thereto, thus exhibiting a dual function inblocking selectin-mediated adhesion due to P-selectin/PSGL-1 binding andin causing dissociation of preformed P-selectin/PSGL-1 complexes.

The screening method in a preferred embodiment comprises in vitro assaysthat can be used to identify anti-P-selectin antibodies that abolishP-selectin/PSGL-1 binding and preferably cause dissociation of preformedP-selectin/PSGL-1 complexes. Test anti-P-selectin antibodies can bescreened for dual function capability with a series of assays such as,but not limited to, those described herein which will identify thoseantibodies that bind to a conformational epitope within amino acids1-35, and more particularly within amino acids 4-23, of P-selectin, andthat block the binding of the PSGL-1 ligand to P-selectin, and whichpreferably cause dissociation of preformed P-selectin/PSGL-1 complexes.No anti-P-selectin antibodies have heretofore been shown to have theability to both block PSGL-1 binding to P-selectin and to causedissociation of preformed P-selectin/PSGL-1 complexes.

In a first step of the screening method, for example, test antibodiesgenerated against P-selectin are assayed for the ability to blockbinding of PSGL-1 to P-selectin. Test antibodies which block binding ofPSGL-1 to P-selectin are screened to determine their ability to causedissociation of preformed P-selectin/PSGL-1 complexes. Test antibodiesidentified as having dual function of blocking both PSGL-1 binding toP-selectin, and causing dissociation of P-selectin/PSGL-1 complexcomprise particularly preferred embodiments of the presently disclosedand claimed inventive concepts and can be used in the methods of thepresently disclosed and claimed inventive concepts.

In one embodiment of the method, test antibodies which block binding ofPSGL-1 to P-selectin are first identified using a first screening assay.For example, in a preferred embodiment of the first screening assay,PSGL-1 or a synthetic PSGL-1 mimetic such as GSP-6, or a terminalepitope portion of PSGL-1 able to bind to P-selectin, is bound to asubstrate, such as a BIACORE chip, in a method known to persons havingordinary skill in the art. The substrate having the PSGL-1 (or thePSGL-1 mimetic) bound thereto is then exposed to an anti-P-selectin testantibody/P-selectin complex. The binding of the complex to thePSGL-1-substrate is then evaluated. If the test antibody/P-selectincomplex does not bind to the PSGL-1 on the substrate, the test antibodyis identified as a “function-blocking” antibody.

In an alternative embodiment of the first screening assay, P-selectin,or a portion thereof which maintains the integrity of the conformationalepitope, is bound to the substrate. For example, the minimum portion ofP-selectin which maintains the conformational epitope comprises thelectin and EGF binding domains of P-selectin. In this embodiment, theP-selectin-substrate is exposed to the test antibody, which binds toform the P-selectin-antibody complex. Then PSGL-1, or a high molecularweight mimetic thereof such as a GSP-6/biotin/avidin complex, is exposedto the P-selectin/test antibody complex and the binding thereto ofPSGL-1 (or the mimetic) is evaluated. An antibody which prevents orinhibits the binding of PSGL-1 to P-selectin is identified as a“function-blocking” antibody. When the GSP-6 or PSGL-1 mimetic is boundto the substrate, in a preferred embodiment, it is bound to biotin, andthe mimetic-biotin complex is bound to a streptavidin coating on thesubstrate.

In a preferred embodiment of the second screening assay, either PSGL-1or a mimetic thereof is bound, as described above, to the substrate(such as a BIACORE chip). P-selectin is then applied to thePSGL-1/substrate to form the P-selectin/PSGL-1 (or mimetic) complex. Thetest antibody is then applied and dissociation of the complex ismeasured as a decrease in mass or as Response Units (RU) sinceP-selectin is being dissociated away. A function-blockinganti-P-selectin antibody, which induces dissociation of theP-selectin/PSGL-1 complex, is designated as a dual functionanti-P-selectin antibody. In yet another embodiment of the second assay,the anti-P-selectin antibody itself is bound to the substrate, and aP-selectin/PSGL-1 complex is exposed to it, and dissociation thereof ismeasured.

In an alternate embodiment of the second screening assay, P-selectin maybe bound to a substrate rather than the PSGL-1. PSGL-1 or a highmolecular weight mimetic thereof such as a GSP-6/biotin/avidin complexis then exposed to the P-selectin on the substrate and allowed to form aPSGL-1/P-selectin complex. The PSGL-1/P-selectin complex is then exposedto a function-blocking anti-P-selectin antibody and dissociation of thecomplex is evaluated, for example using a BIACORE method as describedelsewhere herein.

Although the presently disclosed and claimed inventive concepts and itsadvantages have been described in detail, it should be understood thatvarious changes, substitutions and alterations can be made hereinwithout departing from the spirit and scope of the presently disclosedand claimed inventive concepts as defined by the appended claims.Moreover, the scope of the present application is not intended to belimited to the particular embodiments of the process, machine, items ofmanufacture, compositions of matter, means, methods and steps describedin the specification. As one having ordinary skill in the art willreadily appreciate from the present disclosure, processes, machines,items of manufacture, compositions of matter, means, methods, or steps,presently existing or later to be developed that perform substantiallythe same function or achieve substantially the same result as thecorresponding embodiments described herein may be utilized according tothe presently disclosed and claimed inventive concepts. Accordingly, theappended claims are intended to include within their scope suchprocesses, machines, items of manufacture, compositions of matter,means, methods, or steps.

Each of the references, patents or publications cited herein isexpressly incorporated herein by reference in its entirety.

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1-37. (canceled)
 38. An isolated antibody or binding fragment thereofwhich specifically binds to a conformational epitope of P-selectin,wherein the conformational epitope is within amino acid positions 1-35of SEQ ID NO:1.
 39. The isolated antibody or binding fragment of claim38 wherein the conformational epitope is within amino acids 4-23 of SEQID NO:1.
 40. The isolated antibody or binding fragment of claim 38wherein the conformational epitope comprises amino acid positions 4, 14,17, 21, and 22 of SEQ ID NO:1.
 41. The isolated antibody or bindingfragment of claim 40 wherein the conformational epitope furthercomprises amino acid positions 20 and 23 of SEQ ID NO:1.
 42. Theisolated antibody or binding fragment of claim 40 wherein the aminoacids in amino acid positions 4, 14, 17, 21, and 22 are H, I, K, N, andR, respectively.
 43. The isolated antibody or binding fragment thereofof claim 40 wherein binding is abrogated when any one or more of aminoacid positions 4, 14, 17, 21, or 22 is substituted with N,N, V, R, or H,respectively.
 44. The isolated antibody or binding fragment of claim 38comprising the ability to block the binding of P-selectin glycoproteinligand-1 (PSGL-1) to P-selectin.
 45. The isolated antibody or bindingfragment of claim 38 further comprising the ability to causedissociation of a preformed P-selectin-PSGL-1 complex.
 46. The isolatedantibody or binding fragment of claim 38 comprising the ability to blockthe function of P-selectin by inhibiting the binding of activatedendothelial cells to leukocytes, lymphocytes, sickled red cells, and/orplatelets.
 47. The isolated antibody or binding fragment of claim 38comprising the ability to block the function of P-selectin by inhibitingthe binding of activated platelets to leukocytes, lymphocytes, sickledred cells, and/or platelets.
 48. The isolated antibody or bindingfragment of claim 38 comprising the ability to cause dissociation ofcell-cell binding between activated endothelial cells and leukocytes,lymphocytes, sickled red cells, and/or platelets.
 49. The isolatedantibody or binding fragment of claim 38 comprising the ability to causedissociation of cell-cell binding between activated platelets andleukocytes, lymphocytes, sickled red cells, and/or platelets.
 50. Theisolated antibody or binding fragment of claim 38 wherein the antibodyor fragment thereof is monoclonal.
 51. The isolated antibody or bindingfragment of claim 38 wherein the antibody or fragment thereof ischimeric, human, or humanized.
 52. The isolated antibody or bindingfragment of claim 38 comprising an immunoglobulin selected from theclass consisting of IgA, IgD, IgE, IgG, and IgM.
 53. The isolatedantibody or binding fragment of claim 52 wherein the isolated antibodyor binding fragment thereof is an IgG selected from an isotypeconsisting of IgG1, IgG2, IgG3, IgG4, or an IgG2/G4 chimera.
 54. Thebinding fragment of claim 38 comprising at least one of a Fab, Fab′,F(ab)₂, or scFv fragment.
 55. The isolated antibody or binding fragmentof claim 38 which binds to the conformational epitope with a K_(d)≦1000nM, a K_(d)≦500 nM, a K_(d)≦100 nM, a K_(d)≦50 nM, a K_(d)≦25 nM, aK_(d)≦10 nM, a K_(d)≦5 nM, a K_(d)≦1 nM, or a K_(d)≦0.1 nM.
 56. Acomposition comprising the isolated antibody or binding fragment ofclaim 38 disposed within a pharmaceutically-acceptable carrier, vehicle,or diluent.
 57. A nucleic acid which encodes the antibody or bindingfragment thereof of claim
 38. 58. A cell line which expresses theantibody or binding fragment of claim 38.